Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications

Abstract The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 Umg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Resumo As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 Umg-1. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These byproducts can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-1. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Exopolysaccharyde production by Cryptococcus laurentii SD7 using molasses and corn steep liquor as substrates

Microbial polysaccharides are of great biotechnological and commercial interest and have wide application in the food, cosmetic and medicine industries. Exopolysaccharide (EPS) production by the yeast Cryptococcus laurentii SD7, isolated from fresh water molluscs, was studied using agro-industrial byproducts as substrates in the submerged fermentation. The Central Composite Design (CCD) 23 was used to study the influence of pH, different concentrations on sugarcane molasses and corn steep liquor (CSL), for 48 hours. According to the results, the highest EPS production occurred at the initial pH 5 and at 8.4% concentration of sugarcane molasses, which were statistically significant variable at 10% (p < 0.1). The concentration of CSL had no influence in the studied range, thus, it can be used lowest concentration (0.3%). The time course of EPS production showed that while cell growth peaked within 48 hours, the highest EPS production (6.61 g L-1) occurred at 168 hours, with a productivity of about 0.04 g L-1 h-1. The pH of the medium remained approximately constant throughout the fermentation process. The yeast C. laurentii SD7 showed great potential for EPS production at a low cost and with sustainable substrates.(AU)

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.(AU)

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.(AU)

Exopolysaccharyde production by Cryptococcus laurentii SD7 using molasses and corn steep liquor as substrates / Exopolysaccharyde production by Cryptococcus laurentii SD7 using molasses and corn steep liquor as substrates

Microbial polysaccharides are of great biotechnological and commercial interest and have wide application in the food, cosmetic and medicine industries. Exopolysaccharide (EPS) production by the yeast Cryptococcus laurentii SD7, isolated from fresh water molluscs, was studied using agro-industrial byproducts as substrates in the submerged fermentation. The Central Composite Design (CCD) 23 was used to study the influence of pH, different concentrations on sugarcane molasses and corn steep liquor (CSL), for 48 hours. According to the results, the highest EPS production occurred at the initial pH 5 and at 8.4% concentration of sugarcane molasses, which were statistically significant variable at 10% (p < 0.1). The concentration of CSL had no influence in the studied range, thus, it can be used lowest concentration (0.3%). The time course of EPS production showed that while cell growth peaked within 48 hours, the highest EPS production (6.61 g L-1) occurred at 168 hours, with a productivity of about 0.04 g L-1 h-1. The pH of the medium remained approximately constant throughout the fermentation process. The yeast C. laurentii SD7 showed great potential for EPS production at a low cost and with sustainable substrates.

Microbial polysaccharides are of great biotechnological and commercial interest and have wide application in the food, cosmetic and medicine industries. Exopolysaccharide (EPS) production by the yeast Cryptococcus laurentii SD7, isolated from fresh water molluscs, was studied using agro-industrial byproducts as substrates in the submerged fermentation. The Central Composite Design (CCD) 23 was used to study the influence of pH, different concentrations on sugarcane molasses and corn steep liquor (CSL), for 48 hours. According to the results, the highest EPS production occurred at the initial pH 5 and at 8.4% concentration of sugarcane molasses, which were statistically significant variable at 10% (p < 0.1). The concentration of CSL had no influence in the studied range, thus, it can be used lowest concentration (0.3%). The time course of EPS production showed that while cell growth peaked within 48 hours, the highest EPS production (6.61 g L-1) occurred at 168 hours, with a productivity of about 0.04 g L-1 h-1. The pH of the medium remained approximately constant throughout the fermentation process. The yeast C. laurentii SD7 showed great potential for EPS production at a low cost and with sustainable substrates.

The importance of physiological quality of seeds for agriculture / A importância da qualidade fisiológica de sementes para a agricultura

Seeds present a fundamental role since the beginnings of agriculture, propelling the agricultural development of different people in different ages of human history. Its importance has been linked to the possibility of domesticating the most diverse plants in the past and, nowadays, of providing many biotechnological advancements represented by the most diverse cultivars and hybrids introduced into the market. However, the expression of all its capacities depends on the quality of this supply represented by the sum of physical, genetic, sanitary, and physiological attributes. This review shows how the physiological component of the quality of seeds has influenced the agricultural process for the most diverse crops, notedly, for major crops, forages, or vegetables. It is highlighted its central role in fulfilling the growing demands o fa growing world population. We emphasize the preoccupation of research, development, and innovation actions in the sense of recognizing the factors that influence the physiological quality of seeds, developing and enhancing methods to estimate, preserve, and increase it, and how the adoption of high physiological quality seeds has influenced the development of the major crops.

As sementes representam papel fundamental desde os primórdios da agricultura, impulsionando o desenvolvimento agrícola dos diferentes povos, em diferentes épocas da História da humanidade. Sua importância esteve ligada à possibilidade de domesticação dos mais diversos cultivos, no passado, e, atualmente, são portadoras de inúmeros avanços biotecnológicos, representados pelas mais diversas cultivares e híbridos lançados no mercado. A expressão de todas as suas potencialidades, porém, depende da qualidade desse insumo, representada pelo somatório de atributos, físicos, genéticos, sanitários e fisiológicos. A presente revisão pretende evidenciar como o componente fisiológico da qualidade de sementes tem influenciado o progresso da agricultura para os mais diferentes cultivos, notadamente, para as espécies de grandes culturas, forrageiras e hortaliças, ressaltando seu protagonismo para o atendimento das crescentes demandas de uma população mundial em constante crescimento. Destaca-se a preocupação das ações de pesquisa, desenvolvimento e inovação no sentido de conhecer os fatores que influenciam a qualidade fisiológica das sementes, desenvolver e aprimorar métodos para estimá-la, preservá-la ou incrementá-la e como a adoção de sementes de elevada qualidade fisiológica tem impactado o desenvolvimento dos principais cultivos.

The importance of physiological quality of seeds for agriculture / A importância da qualidade fisiológica de sementes para a agricultura

Seeds present a fundamental role since the beginnings of agriculture, propelling the agricultural development of different people in different ages of human history. Its importance has been linked to the possibility of domesticating the most diverse plants in the past and, nowadays, of providing many biotechnological advancements represented by the most diverse cultivars and hybrids introduced into the market. However, the expression of all its capacities depends on the quality of this supply represented by the sum of physical, genetic, sanitary, and physiological attributes. This review shows how the physiological component of the quality of seeds has influenced the agricultural process for the most diverse crops, notedly, for major crops, forages, or vegetables. It is highlighted its central role in fulfilling the growing demands o fa growing world population. We emphasize the preoccupation of research, development, and innovation actions in the sense of recognizing the factors that influence the physiological quality of seeds, developing and enhancing methods to estimate, preserve, and increase it, and how the adoption of high physiological quality seeds has influenced the development of the major crops.(AU)

As sementes representam papel fundamental desde os primórdios da agricultura, impulsionando o desenvolvimento agrícola dos diferentes povos, em diferentes épocas da História da humanidade. Sua importância esteve ligada à possibilidade de domesticação dos mais diversos cultivos, no passado, e, atualmente, são portadoras de inúmeros avanços biotecnológicos, representados pelas mais diversas cultivares e híbridos lançados no mercado. A expressão de todas as suas potencialidades, porém, depende da qualidade desse insumo, representada pelo somatório de atributos, físicos, genéticos, sanitários e fisiológicos. A presente revisão pretende evidenciar como o componente fisiológico da qualidade de sementes tem influenciado o progresso da agricultura para os mais diferentes cultivos, notadamente, para as espécies de grandes culturas, forrageiras e hortaliças, ressaltando seu protagonismo para o atendimento das crescentes demandas de uma população mundial em constante crescimento. Destaca-se a preocupação das ações de pesquisa, desenvolvimento e inovação no sentido de conhecer os fatores que influenciam a qualidade fisiológica das sementes, desenvolver e aprimorar métodos para estimá-la, preservá-la ou incrementá-la e como a adoção de sementes de elevada qualidade fisiológica tem impactado o desenvolvimento dos principais cultivos.(AU)

Xanthan gum production by Xanthomonas axonopodis pv. mangiferaeindicae from glycerin of biodiesel in different media and addition of glucose

Biodiesel production has been increasing yearly in Brazil. A large amount of glycerin is generated in this process and needs a correct destination. One possible use of this glycerin in crude form is in biotechnological processes. Xanthan gum is a commercial gum used primarily in the pharmaceutical and food industries as thickener, emulsifier and stabilizer. It is synthetized by species of the bacterium Xanthomonas generally from glucose. However, current research shows that species of this bacterium have the capacity to grow and synthesize the gum using glycerin from biodiesel. The aim of this study was to produce xanthan gum using glycerin from biodiesel production in medium with different nitrogen content, named complex and simple media. The kinetics of fermentation in simple medium showed a twofold increase in gum production (3.16 kg.m-3) compared to the one in complex medium (1.46 kg.m-3) after 120 hours. The gum generated in this study showed chemical and rheological characteristics of xanthan gum. Glucose supplementation did not show an increase in xanthan production but did increase the consistency index and the behavioral index of solutions of this gum.

Xanthan gum production by Xanthomonas axonopodis pv. mangiferaeindicae from glycerin of biodiesel in different media and addition of glucose

Biodiesel production has been increasing yearly in Brazil. A large amount of glycerin is generated in this process and needs a correct destination. One possible use of this glycerin in crude form is in biotechnological processes. Xanthan gum is a commercial gum used primarily in the pharmaceutical and food industries as thickener, emulsifier and stabilizer. It is synthetized by species of the bacterium Xanthomonas generally from glucose. However, current research shows that species of this bacterium have the capacity to grow and synthesize the gum using glycerin from biodiesel. The aim of this study was to produce xanthan gum using glycerin from biodiesel production in medium with different nitrogen content, named complex and simple media. The kinetics of fermentation in simple medium showed a twofold increase in gum production (3.16 kg.m-3) compared to the one in complex medium (1.46 kg.m-3) after 120 hours. The gum generated in this study showed chemical and rheological characteristics of xanthan gum. Glucose supplementation did not show an increase in xanthan production but did increase the consistency index and the behavioral index of solutions of this gum.(AU)

Drying of microalga Scenedesmus obliquus BR003 in a gas dryer at low temperatures / Secagem da microalga Scenedesmus obliquus BR003 em secador a gás em baixas temperaturas

O desenvolvimento de equipamentos eficientes e específicos para a secagem de microalgas é essencial para a exploração comercial destes microrganismos que apresentam alta taxa de crescimento e grande potencial biotecnológico. Os custos de secagem da biomassa de microalgas ainda são elevados e precisam ser reduzidos para a produção de compostos com baixo valor agregado. Portanto, realizou-se o estudo da secagem da microalga Scenedesmus obliquus BR003 utilizando baixas temperaturas. S. obliquus BR003 é uma microalga robusta que apresenta alta produtividade de lipídeos. Em escala laboratorial, observou-se que a biomassa de S. obliquus BR003 foi rapidamente seca em baixas temperaturas entre 50 e 60 ºC. Um secador a gás foi utilizado para avaliar a secagem da biomassa de S. obliquus BR003 em escala piloto. A biomassa foi seca em menos de 24 h utilizando o secador a gás, entretanto, a elevada umidade da biomassa da microalga requereu uma maior renovação de ar na câmara do secador. A análise de fluidodinâmica computacional do secador a gás mostrou dois parâmetros importantes para se obter uma maior efetividade de transferência de calor e massa durante o processo de secagem da biomassa de microalga. Concluiu-se que um secador a gás adequado, para a biomassa de microalgas, deve possuir múltiplos pontos de injeção de ar, e um eficiente sistema de circulação e renovação de ar no interior da câmara de secagem.(AU)

Development of efficient and specific equipment to dry microalgae is essential for commercial use of these microorganisms that show high growth rates and biotechnological potential. Drying costs of microalgae biomass are still high and they should be reduced for the production of compounds with low added value. Therefore, we evaluated the drying process of the microalga Scenedesmus obliquus BR003 using low temperatures. S. obliquus BR003 is a robust microalga that shows high lipid productivity. At laboratory scale, it was observed that the biomass of S. obliquus BR003 was rapidly dried at low temperatures between 50 and 60 ° C. A gas dryer was used to evaluate the drying of the biomass of S. obliquus BR003 on a pilot-scale. The biomass was dried in less than 24 h using the gas dryer; however, the high moisture of the microalga biomass required a higher air renovation in the drying chamber. Computational fluid dynamics analysis of the gas dryer showed two important parameters to achieve greater effectiveness of heat and mass transfer rates during the drying process of the microalga biomass. It was concluded that a gas dryer suitable for the microalgae biomass should have multiple air injection points, and an efficient circulation and renovation system of air inside the drying chamber.(AU)

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

Background:The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells.Methods:BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array.Results:BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05).Conclusion:Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells...(AU)

Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil

Carnivorous plant species, such as Utricularia spp., capture and digest prey. This digestion can occur through the secretion of plant digestive enzymes and/or by bacterial digestive enzymes. To comprehend the physiological mechanisms of carnivorous plants, it is essential to understand the microbial diversity related to these plants. Therefore, in the present study, we isolated and classified bacteria from different organs of Utricularia breviscapa (stolons and utricles) and from different geographic locations (São Paulo and Mato Grosso). We were able to build the first bacterium collection for U. breviscapa and study the diversity of cultivable bacteria. The results show that U. breviscapa bacterial diversity varied according to the geographic isolation site (São Paulo and Mato Grosso) but not the analyzed organs (utricle and stolon). We reported that six genera were common to both sample sites (São Paulo and Mato Grosso). These genera have previously been reported to be beneficial to plants, as well as related to the bioremediation process, showing that these isolates present great biotechnological and agricultural potential. This is the first report of an Acidobacteria isolated from U. breviscapa. The role of these bacteria inside the plant must be further investigated in order to understand their population dynamics within the host.(AU)

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 8h.(AU)

Current applications and different approaches for microbial L-asparaginase production

ABSTRACT L-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in L-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, L-asparaginase catalyzes the hydrolysis of L-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of L-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents L-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production.

Current applications and different approaches for microbial L-asparaginase production

L-asparaginase (EC 3.5.1.1) is an enzyme that catalysis mainly the asparagine hydrolysis in L-aspartic acid and ammonium. This enzyme is presented in different organisms, such as microorganisms, vegetal, and some animals, including certain rodent's serum, but not unveiled in humans. It can be used as important chemotherapeutic agent for the treatment of a variety of lymphoproliferative disorders and lymphomas (particularly acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma), and has been a pivotal agent in chemotherapy protocols from around 30 years. Also, other important application is in food industry, by using the properties of this enzyme to reduce acrylamide levels in commercial fried foods, maintaining their characteristics (color, flavor, texture, security, etc.) Actually, L-asparaginase catalyzes the hydrolysis of L-asparagine, not allowing the reaction of reducing sugars with this aminoacid for the generation of acrylamide. Currently, production of L-asparaginase is mainly based in biotechnological production by using some bacteria. However, industrial production also needs research work aiming to obtain better production yields, as well as novel process by applying different microorganisms to increase the range of applications of the produced enzyme. Within this context, this mini-review presents L-asparaginase applications, production by different microorganisms and some limitations, current investigations, as well as some challenges to be achieved for profitable industrial production.(AU)

Kluyveromyces lactis beta-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose / Imobilização da beta-galactosidase de Kluyveromyces lactis em esferas de alginato de cálcio e gelatina para hidrólise da lactose presente no soro de queijo

One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.

Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.

Kluyveromyces lactis beta-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose / Imobilização da beta-galactosidase de Kluyveromyces lactis em esferas de alginato de cálcio e gelatina para hidrólise da lactose presente no soro de queijo

One of the greatest challenges for dairy industries is the correct destination of all the whey generated during cheese making, considering its high impact, the large volume created, and its technological potential. Enzymatic hydrolysis of cheese whey lactose is a biotechnological alternative. However, one of the limiting factors of its use is the relatively high cost of the enzymes, which could be lowered with the immobilization of these biocatalysts. Considering this context, the objective of this research was to evaluate the commercial Kluyveromyces lactis -galactosidase enzyme immobilized in calcium alginate spheres and gelatin, using glutaraldehyde and concanavalin A (ConA) as modifying agents in the hydrolysis of cheese whey lactose process. Results have shown that the enzyme encapsulation complexed with ConA in alginate-gelatin spheres, without glutaraldehyde in the immobilization support, has significantly increased the hydrolysis of lactose rate, achieving a maximum conversion of 72%.(AU)

Um dos grandes desafios das indústrias de laticínios é destinar de forma correta todo o soro gerado durante a produção de queijo, devido ao seu impacto ambiental, grande volume gerado e potencial tecnológico. A hidrólise enzimática da lactose presente no soro de queijo é uma alternativa biotecnológica. Contudo, um dos fatores limitantes de sua utilização é o custo relativamente alto das enzimas, o que poderia ser minimizado com a imobilização destes biocatalisadores. Baseado nesse contexto, o objetivo do presente trabalho foi avaliar a enzima comercial -galactosidase de Kluyveromyces lactis , imobilizada em esferas de alginato de cálcio e gelatina, empregando o glutaraldeído e a concanavalina A (ConA) como agentes modificadores, no processo de hidrólise da lactose presente no soro de queijo. Os resultados obtidos demonstraram que o encapsulamento da enzima complexada com ConA em esferas de alginato-gelatina, sem a presença de glutaraldeído no meio de imobilização, aumentou de modo significativo o teor de hidrólise da lactose, obtendo conversão máxima de 72%.(AU)

CARACTERIZAÇÃO ESTRUTURAL DA LECTINA DA ESPONJA MARINHA Chondrilla caribensis

Os organismos marinhos possuem uma grande diversidade química, sendo capazes de produzir inúmeros compostos diferentes. Dentre esses compostos estão as lectinas, proteínas ou glicoproteínas que apresentam ligação específica e reversível a carboidratos, porém não alteram as propriedades e nem estão envolvidas necessariamente ao metabolismo dos mesmos. O estudo das lectinas e de suas interações proteína-carboidrato são de grande importância, já que o padrão de glicosilação dos componentes celulares é influenciado por alterações fisiológicas, como a ocorrência de doenças. Já os estudos estruturais são relevantes para solucionar e entender o papel fisiológico dentro do organismo e avaliar seu potencial biotecnológico. Apesar dos estudos estruturais de lectinas terem sido impulsionados há muito tempo, poucas lectinas de esponjas possuem sua estrutura primária determinada e apenas uma tem sua estrutura tridimensional resolvida. O objetivo deste trabalho foi caracterizar estruturalmente uma lectina presente na esponja marinha Chondrilla caribensis e avaliar o potencial da mesma em diferentes atividades biológicas. A estrutura primária de CCL foi determinada por sobreposição de peptídeos sequenciados por espectrometria de massas. A sequência de aminoácidos da proteína consiste de 142 aminoácidos com massa molecular calculada de 15.443 Da. A lectina possui uma arquitetura de domínio do tipo galectina, família de lectinas ligantes a -galactosídeos, sendo esta família bastante difundida no reino animal. A sequência assinatura de domínio altamente conservado em galectinas foi identificada em CCL com algumas modificações observadas também em outras galectinas de esponjas. A estrutura tridimensional foi prevista e o local de ligação a carboidratos analisado por meio do docking molecular, onde CCL exibe uma estrutura típica das galectinas que consiste em um -sanduíche com duas -folhas antiparalelas. Os aminoácidos que fazem interações com os ligantes de CCL no sítio de ligação ao monossacarídeo são, na maioria, os mesmos conservados nesta família de lectinas. A função molecular, o processo biológico e a localização da proteína prevista, mostram que CCL é uma típica galectina podendo desempenhar várias funções diferentes tanto intracelular como extracelular. CCL é uma molécula com potencial antitumoral contra células de carcinoma de próstata e adenocarcinoma de mama, com baixa citotoxicidade contra células saudavéis, além de mostrar atividade leishmanicida com resultados bastante promissores.

The organisms have a great chemical diversity, being able to produce countless diferente compounds. Among these compounds are lectins, proteins or glycoproteins that have specific and reversible binding to carbohydrates, however they do not alter their properties and are not necessarily involved in their metabolismo. The study of lectins and their protein-carbohydrate interactions are of great importance, since the pattern of glycosylation of cellular components is influenced by physiological changes, such as the occurrence of diseases. Structural studies are relevant to solve and understand the physiological role within the organismo and to evaluate its biotechnological potential. Although structural studies of lectins have been promoted for a long time, few sponges lectins have their primary strucuture determined and only one of them has its three-dimensional structure resolved. The objective of this work was to structurally characterize the lectin presente in the marine sponge Chondrilla caribensis and to evaluate its potential in diferente biological activities. The primary structure of CCL was determined by mass spectrometry. The amino acid sequence of the protein consists of 142 amino acids with a calculated molecular mass of 15.443 Da. Lectin has a galectin-like domain architecture, a Family of -galactoside-binding lectins, and is very widespread in the animal kingdom. The signature sequence of highly conserved domain in galectins was identified in CCL with some modifications observed also in other sponge galectins. The three-dimensional structure was predicted and the carbohydrate binding site was analyzed using molecular docking, where CCL exhibits a typical galectin structure consisting of a -sandwich with two antiparallel -sheets. The amino acids that interact with the CCL ligands at the monosaccharide binding site are mostly the same conserved in this Family of lectins. The molecular function, the biological process and the location of the predicted protein, show that CCL is a typical galectin and can perform several diferente functions, both intracelular and extracelular. CCL is a molecule with antitumor potential against prostate carcinoma cells and breast adenocarcinoma, with low cytotoxicity against healthy cells, in addition to showing leishmanicidal activity with very promising results.

IMOBILIZAÇÃO DE PROTEASES COLAGENOLÍTICAS OBTIDAS DE Aspergillus sclerotiorum URM 5792 EM NANOPARTÍCULAS MAGNÉTICAS REVESTIDAS COM QUITOSANA

As proteases são enzimas quem têm sido extensivamente utilizadas em diversos segmentos da indústria. Apesar de apresentar elevado interesse e inúmeras vantagens, sua aplicação expõe ainda algumas limitações. O processo de imobilização tem se tornado uma alternativa, visto que pode aumentar a estabilidade enzimática e promover a reutilização por vários ciclos. A utilização de suportes magnéticos tem sido amplamente explorada, devido a sua versatilidade no setor da biotecnologia de purificação de biomoléculas, e seu revestimento com suportes orgânicos como a quitosana, facilitam o processo, impedindo a oxidação das nanopartículas magnéticas (NPMs), conferindo propriedades ideais para a imobilização. O presente trabalho teve como objetivo selecionar, imobilizar e caracterizar proteases colagenolíticas obtidas de Aspergillus scletotiorum URM5792 em NPMs revestidas com quitosana. Para produção foi realizado um planejamento fatorial completo 22; tendo como melhor condição de produção (7g de farelo de trigo e 60% de umidade, submetidas a 30°C por 72h de fermentação, com atividade proteolítica de 56,27 U/mL e colagenolítica de 303,00 U/mL). O processo de imobilização foi realizado utilizando planejamento fatorial completo 23 visando avaliar a influência das variáveis independentes: concentração de glutaraldeído, tempo de ativação e tempo de imobilização sob o rendimento de imobilização enzimática. No ensaio composto por 4% de concentração de glutaraldeído, 2,5h de ativação e 1,5h de imobilização foram obtidos rendimentos de 86,25% para atividade proteásica e 83,83% para atividade colagenolítica. A enzima imobilizada apresentou mais de 95% da atividade inicial após 28 dias de armazenamento e reteve mais de 60% da atividade residual no décimo segundo ciclo de reutilização. Também foram analisadas a influência do pH e temperatura sob a atividade enzimática na forma livre e imobilizada, ambas apresentaram pH ótimo na faixa de 9,0, assim como, temperatura ótima de 30°C e 40°C, respectivamente. A estabilidade ao pH e à temperatura da enzima livre se manteve com mais de 80% e 60% de atividade residual nos tempos de 24h e 180min, respectivamente. E a estabilidade da enzima imobilizada se manteve com 60% e 70% de atividade residual nos mesmos tempos. A fermentação em estado sólido foi eficaz com alta produção enzimática e a imobilização da protease colagenolítica em nanopartículas magnéticas revestidas com quitosana e ativadas em glutaraldeído, mostraram ser métodos eficientes no rendimento, armazenamento e reuso da enzima. A enzima imobilizada apresentou maior afinidade ao substrato em relação à enzima livre, foi inibida pelo íon Cu2+, SDS e PMSF indicando a presença de uma serino-protease. Esses resultados indicam que Aspergillus sclerotiorum URM 5792 é uma fonte potencial para a produção de protease colagenolítica com possíveis aplicações biotecnológicas em diversos setores da indústria, na produção de detergentes, no setor têxtil e na indústria farmacêutica, no tratamento e regeneração de tecidos em necrose.

Proteases are enzymes that have been used extensively in several industry segments. Despite their high interest and numerous advantages, their application still has some limitations. The immobilization process has become an alternative since it increases enzyme stability. The use of magnetic supports has been widely explored, due to its versatility in the biotechnology sector for the purification of biomolecules, and its coating with organic supports such as chitosan, facilitates the process, preventing the oxidation of magnetic nanoparticles (MNP's), giving ideal properties for immobilization. The present work aimed to select, immobilize and characterize collagenolytic proteases obtained from Aspergillus sclerotiorum URM5792 in MNPs coated with chitosan. For production, a complete factorial planning was carried out 22; with the best production condition (7g of wheat bran and 60% humidity, submitted to 30°C for 72h of fermentation, with proteolytic activity of 56,27 U/mL and e collagenolytic of 303,00 U/mL). The immobilization process was carried out using complete factorial planning 23 aiming to evaluate the influence of independent variables: glutaraldehyde concentration, activation time and immobilization time under the enzyme immobilization yield. In the assay composed of 4% glutaraldehyde concentration, 2.5h of activation and 1.5h of immobilization yields of 86.25% for protein activity and 83.83% for collagenolytic activity were obtained. The immobilized enzyme showed more than 95% of the initial activity after 28 days of storage and retained more than 60% of the residual activity in the twelfth cycle of reuse. The influence of pH and temperature on enzyme activity in free and immobilized form were also analyzed, both had an optimal pH in the range of 9.0, as well as an optimal temperature of 30 ° C and 40 ° C, respectively. The pH and temperature stability of the free enzyme was maintained with more than 80% and 60% of residual activity within 24 hours and 180 minutes, respectively. And the stability of the immobilized enzyme was maintained with 60% and 70% of residual activity in the same times. The solid-state fermentation was effective with high enzymatic production and the immobilization of protease collagenolytic in magnetic nanoparticles coated with chitosan and activated in glutaraldehyde, proved to be efficient methods in the yield, storage and reuse of the enzyme. The immobilized enzyme showed greater affinity to the substrate in relation to the free enzyme, it was inhibited by the Cu2+ ion, SDS and PMSF indicating the presence of active serine protease sites. These results indicate that Aspergillus sclerotiorum URM 5792 is a potential source for production of collagenolytic protease with possible biotechnological applications in various sectors of the industry, in the production of detergents, in the textile and pharmaceutical industry, in the treatment and regeneration of tissues in necrosis.