Expression of antigens of foot-and-mouth disease virus in plants: a review / 生物工程学报

Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV), which seriously endangers the development of animal husbandry. The inactivated FMD vaccine is the main product for the prevention and control of FMD, which has been successfully applied to control the pandemic and outbreak of FMD. However, the inactivated FMD vaccine also has problems, such as the instability of antigen, the risk of spread of the virus due to incomplete inactivation during vaccine production, and the high cost of production. Compared with traditional microbial and animal bioreactors, production of antigens in plants through transgenic technology has some advantages including low cost, safety, convenience, and easy storage and transportation. Moreover, since antigens produced from plants can be directly used as edible vaccines, no complex processes of protein extraction and purification are required. But, there are some problems for the production of antigens in plants, which include low expression level and poor controllability. Thus, expressing the antigens of FMDV in plants may be an alternative mean for production of FMD vaccine, which has certain advantages but still need to be continuously optimized. Here we review the main strategies for expressing active proteins in plants, as well as the research progress on the expression of FMDV antigens in plants. We also discuss the current problems and challenges encountered, with the aim to facilitate related research.

Advances in research and application of animal cell culture matrix fibracel carriers / 中国生物制品学杂志

@#Fibracel carriers based on polyester fiber have the advantages of good acid and alkali resistance,heat resistance,good biocompatibility,non-biodegradation,and can promote cell adhesion and growth. With no animal-derived ingredients and high biological safety,it is one of the preferred carriers for cell culture matrixes,which has been widely used in the development and production of cell matrix biological products with the development of biotechnology in recent years. This paper reviewed the structure and cell culture characteristics of fibracel carriers as well as the applications in vaccine production,cell therapy and tissue engineering,so as to provide a theoretical basis for the further development and application of fibracel carrier technology.

Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Rational design of a 500 m3 fermenter for erythromycin production by Saccharopolyspora erythraea / 生物工程学报

Erythromycin is a macrolide antibiotic produced by Saccharopolyspora erythraea. Its yield is greatly affected by the fermentation conditions and the bioreactor configurations. In this study, a novel scale-up method for erythromycin fermentation was developed based on computational fluid dynamics (CFD) and time constant analysis. Firstly, the dissolved oxygen (DO) was determined as a key parameter according to the physiological properties of S. erythraea cultivated in a 50 L bioreactor. It was found that the time constant of oxygen supply (tmt) in a 500 m3 bioreactor should be less than 6.25 s in order to satisfy the organism's oxygen uptake rate (OUR). Subsequently, a 500 m3 bioreactor was designed using the time constant method combined with empirical correlations. The impeller combination with one BDT8 impeller at bottom and two MSX4 impellers at upper part was determined, and then validated by numerical simulation. The results indicated that the tmt of the bioreactor (< 6.25 s) and the fluid properties, including gas hold-up, shear stress and fluid vector, met the requirements of erythromycin fermentation. Finally, the industrial production of erythromycin in the 500 m3 showed the design method was applicable in large scale fermentation.

Research progress of red blood cells cultured in vitro / 中国输血杂志

Red blood cells (RBCs) are one of the most needed blood products in clinical blood transfusion treatment. At present, RBCs are mainly provided by blood donors, and the contradiction between supply and demand is extremely prominent. Customizable RBCs cultured in vitro bring dawn to solve this contradiction. RBCs can be differentiated and cultured from human induced pluripotent stem cells, immortalized adult erythroid progenitor cells, embryonic stem cells (ESCs), hematopoietic stem cells or the mononuclear cells extracted from umbilical cord blood and peripheral blood. Each method, with its own advantages and drawbacks, are still in the stage of laboratory research. However, according to the current progress, it is not far from clinical application. This paper summarizes the progress in this field.

Exploration of new effective fibrinolytic agent from Alcaligenes aquatilis PJS_1 from polluted soil

Aim: To isolate and identify Alcaligenes aquatilis PJS_1 from slaughter house soil samples for production of enzymatic fibrinolytic agent productionMethodology: Fibrinolytic enzyme producing bacterium was isolated from slaughter house soil samples and identified by biochemical tests and 16S rRNA sequencing. The fibrinolytic enzyme production media was optimized by various factors like energy sources, pH and temperature. Bioreactor used in the experiment was designed with suitable parameters for effective production and purification is by gel filtration chromatography. Blood clotting assay was performed to determine its anticoagulant property. Results: The isolated enzyme producing bacterium was identified as Alcaligenes aquatilis PJS_1. The medium with fructose and urea at pH 7.0 was found to have optimum production when incubated for 24 hr at 37ºC. The crude enzyme was purified by acetone precipitation followed by gel filtration chromatography. The enzyme showed a final specific activity of 629.32 Umg-1 with of 88.24% yield Interpretation: The present study provides information that the enzyme produced by Alcaligenes aquatilis PJS_1 acts as an effective fibrinolytic agent

Avaliação do desempenho de manta geotêxtil em reator em batelada sequencial no tratamento de esgoto sanitário / Performance evaluation of the geotextile blanket in sequential batch reactor in wastewater treatment

RESUMO No presente trabalho, foi avaliado o desempenho de um sistema de lodo ativado em batelada, com módulo de filtração, proporcionado por uma manta geotêxtil, na remoção de matéria orgânica presente em esgoto sanitário. O reator em escala piloto tinha volume útil de 85 L e foi operado durante 182 dias, com tempo total de ciclo de 24 horas de duração e idade do lodo (θc) de 25 dias. O sistema foi composto de um reator convencional (SB1), utilizado como controle e operado nas mesmas condições que o SM1, o qual utilizou a filtração em manta geotêxtil. Para confecção dos três módulos de filtração, foi utilizada uma manta sintética não tecida combinada com três tipos de espaçadores: tela antiderrapante em poliéster com revestimento em PVC (etapa I) durante 56 dias, geomanta tridimensional de filamentos de polipropileno termosoldados (etapa II) durante 49 dias e manta acrílica 100% poliéster (etapa III) durante 76 dias. A concentração de oxigênio dissolvido (OD) manteve-se na faixa de 3 mgO2.L-1, a relação alimento (A)/microrganismo (M) variou de 0,2 a 0,1 gDBO5.gSSV-1.d-1, e a carga orgânica média aplicada mudou de 0,194 a 0,267 kgDQO.m-3.d.-1. A remoção da turbidez manteve-se na faixa de 97%, a de matéria orgânica em termos de demanda bioquímica de oxigênio (DBO5) foi de 93% e entre 87 e 93% para o parâmetro demanda química de oxigênio (DQO), com o reator SM1 apresentando valores de eficiência e estabilidade operacional superiores ao SB1.

ABSTRACT This work evaluated the performance of a batch activated sludge system with a filtration module, provided by a geotextile blanket to remove organic matter present in sewage. The pilot-scale reactor had a useful volume of 85L, and it was operated for 182 days, with a total cycle time of 24 hours, and sludge age (θc) 25 days. The system was based on a conventional controller (SB1), used as a control and operated under the same conditions as the reactor (SM1) using geotextile blanket filtration. The preparation of the three filtration modules was applied to a non-woven synthetic blanket combined with three types of spacers: PVC-coated non-slip polyester mesh (Step I) for 56 days, three-dimensional geo-mat of thermosolded polypropylene filament (Step II) for 49 days, and 100% polyester acrylic blanket (Step III) for 76 days. The OD concentration remained in the range of 3 mgO2L-1, an F/M ratio ranging from 0.2 to 0.1 gBOD5. gVSS-1d-1 and the organic loading rate average applied ranged from 0.194 to 0.267 kg COD / m3.d.-1. Turbidity removal remained in the range of 97%, and the organic matter removal in terms of BOD5 was 93%, and from 87 to 93% in terms of COD, with SM1 reactor showing efficiency and stability values higher than SB1.

Influence of Oxygen Supply on Growth and Laccases Production by Pleurotus sajor-caju PS-2001 in Submerged Process

Abstract (1) Background: Oxygen supply is an important parameter to be considered in submerged cultures. This study evaluated the influence of different conditions for dissolved oxygen (DO) concentration on laccases activities and growth of Pleurotus sajor-caju PS-2001 in submerged process in stirred-tank bioreactor. (2) Methods: Initially, three different conditions were tested: uncontrolled DO and minimum levels of 30% and 80% of saturation, with the pH controlled between 4.5 and 7.0. (3) Results: Best results were observed at 30% DO (26 U mL-1 of laccases at 96 h), whereas higher mycelial biomass was observed at 30% and 80% DO (above 4.5 g L-1). Four different conditions of DO (uncontrolled, 10%, 30% and 50% of saturation) were tested at pH 6.5, with higher laccases activity (80 U mL-1 at 66 h) and lower mycelial growth (1.36 g L-1 at 90 h) being achieved with DO of 30%. In this test, the highest values for volumetric productivity and specific yield factor were determined. Under the different pH conditions tested, the production of laccases is favoured at DO concentration of 30% of saturation, while superior DO levels favours fungal growth. (4) Conclusion: The results indicate that dissolved oxygen concentration is a critical factor for the culture of P. sajor-caju PS-2001 and has important effects not only on laccases production but also on fungal growth.

Application of micro- and mini-bioreactors in biomedicine development and production / 生物工程学报

Micro- and mini-bioreactors are characterized by their miniature working volume and comprehensive monitoring of process data, e.g., biomass, pH, dissolved oxygen, and fluorescence that are on par with conventional bench-top systems. The technical advancements of micro- and mini-bioreactors are supported by single-use material and micro-manufacturing, non-invasive optical sensors, automation such as industrial robotics and the integration of design of experiment software with data acquisition and process control. Owing to the miniature scales, micro-bioreactors typically feature lower turbulence intensity and energy dissipation rate, resulting in different mass transfer, mixing and shear conditions as compared to industrial scale equipment. Mini-bioreactors, nevertheless, are closer to large vessels. Micro- and mini-bioreactors are used mostly in screening and process development nowadays, owing to their combined high throughput and richness of data. They are also the hardware that will enable "precision medicine" in the near future.

3D printing technology and tissue engineering technology in tracheal replacement: Application and hotspot research / 中国组织工程研究

BACKGROUND: Functional tracheal reconstruction remains a surgical challenge due to the lack of satisfactory tracheal substitutes. OBJECTIVE: To review the research hotspot, clinical application, and main obstacles of tissue-engineered trachea METHODS: A computer-based search of PubMed, Medline, and WanFang databases was performed to retrieve relevant articles published from 2004 to 2019 with the search terms “3D printing, tissue-engineered trachea, trachea reconstruction, tracheal replacement” in English and Chinese. A total of 47 literatures were included in the final analysis. RESULTS AND CONCLUSION: At present, the methods of tracheal reconstruction mainly include artificial tracheal transplantation, allotransplantation, autologous tissue transplantation and tissue-engineered tracheal transplantation. Artificial trachea transplants often fail due to rupture, infection and narrowing of the trachea. Allotransplantation requires long-term immunosuppressive therapy, and death is often caused by necrosis and infection because of insufficient angiogenesis after transplantation. Autogenous tissue has limited ability to replicate the structure and function of the trachea and also has surgical trauma. Tissue-engineered trachea can simulate the biological structure and function similar to natural trachea by selecting suitable scaffold materials and implanting seed cells evenly in the scaffold. It seems to be an ideal tracheal substitute. An intact tracheal scaffold was prepared with biodegradable material using 3D printing technology combined with tissue engineering technology and then implanted into the tissue-engineered trachea cultured with mesenchymal stem cells. This provides a new approach to long-segment tracheal defect reconstruction.

Numerical simulation and optimization of impeller combination used in stirred bioreactor / 生物工程学报

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.

Evaluación de producción de biogás y reducción de carga orgánica de vinazas mediante digestión anaerobia / Biogas production evaluation and reduction of vinasse organic load by anaerobic digestion

RESUMEN La vinaza es un residuo derivado de la producción de alcohol carburante, que posee elevada carga orgánica disuelta (46%), bajo pH que oscila entre 3-5 y alta demanda química de oxigeno (DQO) de 200-300 mgDQO/L, dichas características hacen de la vinaza un potencial contaminante. Hoy en día, se han buscado métodos que contribuyan a un adecuado manejo de este residuo, como su aprovechamiento para generar energía. En esta investigación se determinó el efecto de la concentración de vinaza y el tipo de lodo inoculante sobre la tasa global de producción de biogás, actividad metanogénica y reducción de carga orgánica durante el proceso de digestión anaerobia. Se utilizaron lodos como fuente de inóculo, provenientes de una PTAR, de una laguna de vinaza, y de laguna de oxidación y enfriamiento; la producción de biogás se cuantificó con la técnica de desplazamiento de líquido, se determinó la actividad metanogénica específica (AME), se determinó la tasa de remoción de DQO y sólidos totales y se aislaron microorganismos metanogénicos. Se encontró que la concentración de 90% de vinaza con lodos de la laguna de oxidación obtuvo los mejores rendimientos, se cuantificó 674.5 mL de biogás durante 1 8 días en un volumen de trabajo de 400 mL y se calculó la AME de 0.2 gDQOCH4/gSSV.d. Se demostró que a partir de la digestión anaerobia reduce un 25.2% de la DQO y 22.4% de sólidos; los resultados del análisis microbiológico permitieron evidenciar la presencia de microorganismos metanogénicos en el biorreactor anaerobio.

ABSTRACT Sugarcane vinasse is a residue derived from the production of fuel alcohol, that has a high dissolved organic load (46%), pH ranging from 3 to 5, and chemical oxygen demand (COD) from 200 to 300 mg/l. These characteristics make of the vinasse a potential contaminant. Therefore, methods have been sought that contribute to an adequate management of this waste, such as its use to generate energy. In this research, the process of anaerobic digestion of sugarcane vinasse and sludge as inoculant was evaluated based on the global rate of biogas production, methanogenic activity and reduction of organic load. Different concentrations of vinasse and different sources of sludge were tested; WWTP biosolids, vinasse sludge, and sludge from oxidation and cooling lagoon. Biogas production was quantified by technique liquid displacement, was determined the specific methanogenic activity (SMA), removal of COD and total solids were also determined, and isolation of methanogenic microorganisms. It was determined that the concentration of 90% of vinasse with sludge from the oxidation and cooling lagoon produced the highest yield; 674.5 mL of biogas was quantified during 18 days in a work volume of 400 mL and the SMA of 0.2 g DQOCH4 / gSSV.d was calculated. It was demonstrated that the anaerobic digestion it reduces 25.2% of COD and 22.4% of solids, the results of the microbiological analysis allowed to demonstrate the presence of methanogenic microorganisms in the anaerobic biorreactor.

Development of biodegradation process for Poly(DL-lactic acid) degradation by crude enzyme produced by Actinomadura keratinilytica strain T16-1

Background: Plastic waste is a serious problem because it is difficult to degrade, thereby leading to global environment problems. Poly(lactic acid) (PLA) is a biodegradable aliphatic polyester derived from renewable resources, and it can be degraded by various enzymes produced by microorganisms. This study focused on the scale-up and evaluated the bioprocess of PLA degradation by a crude microbial enzyme produced by Actinomadura keratinilytica strain T16-1 in a 5 L stirred tank bioreactor. Results: PLA degradation after 72 h in a 5 L bioreactor by using the enzyme of the strain T16-1 under controlled pH conditions resulted in lactic acid titers (mg/L) of 16,651 mg/L and a conversion efficiency of 89% at a controlled pH of 8.0. However, the PLA degradation process inadvertently produced lactic acid as a potential inhibitor, as shown in our experiments at various concentrations of lactic acid. Therefore, the dialysis method was performed to reduce the concentration of lactic acid. The experiment with a dialysis bag achieved PLA degradation by weight loss of 99.93%, whereas the one without dialysis achieved a degradation of less than approximately 14.75%. Therefore, the dialysis method was applied to degrade a commercial PLA material (tray) with a conversion efficiency of 32%, which was 6-fold more than that without dialysis. Conclusions: This is the first report demonstrating the scale-up of PLA degradation in a 5 L bioreactor and evaluating a potential method for enhancing PLA degradation efficiency.

Study of capability of nanostructured zero-valent iron and graphene oxide for bioremoval of trinitrophenol from wastewater in a bubble column bioreactor

BACKGROUND: Bioremoval of phenolic compounds using fungi and bacteria has been studied extensively; nevertheless, trinitrophenol bioremediation using modified Oscillatoria cyanobacteria has been barely studied in the literature. RESULTS: Among the effective parameters of bioremediation, algal concentration (3.18 g·L−1 ), trinitrophenol concentration (1301 mg·L−1 ), and reaction time (3.75 d) were screened by statistical analysis. Oscillatoria cyanobacteria were modified by starch/nZVI and starch/graphene oxide in a bubble column bioreactor, and their bioremoval efficiency was investigated. Modifiers, namely, starch/zero-valent iron and starch/GO, increased trinitrophenol bioremoval efficiency by more than 10% and 12%, respectively, as compared to the use of Oscillatoria cyanobacteria alone. Conclusions: It was found that starch/nano zero-valent iron and starch/GO could be applied to improve the removal rate of phenolic compounds from the aqueous solution.

Mechanistic role of perfusion culture on bone regeneration

Bone tissue engineering (BTE) aims to develop engineered bone tissue to substitute conventional bone grafts. To achievethis, culturing the cells on the biocompatible three-dimensional (3D) scaffold is one alternative approach. The newfunctional bone tissue regeneration could be feasible by the synergetic combinations of cells, biomaterials and bioreactors.Although the field of biomaterial design/development for BTE applications attained reasonable success, development ofsuitable bioreactor remains still a major challenge. Tissue engineering bioreactors provide the microenvironment requiredfor neo-tissue regeneration, and also can be used to study the physio-chemical cues effect on cell proliferation anddifferentiation in order to produce functional tissue. In this direction, various bioreactors have been developed andevaluated for the successful development of engineered bone tissue. Continues assessment of tissue development andlimitations of the bioreactors lead to the progression of perfusion flow bioreactor system. Improvements in perfusion reactorsystem were able to yield multiple tissue engineered constructs with uniform cell distribution, easy to operate protocols andalso effectively handled for the functional tissue development to meet the adequate supply of engineered graft for clinicalapplication.

Influência da idade do lodo na colmatação das membranas em um biorreator à membrana tratando esgoto sanitário / Effect of solids retention time on membrane fouling in a membrane bioreactor treating municipal wastewater

RESUMO O presente estudo avaliou o efeito da idade do lodo (θc) no potencial incrustante do licor misto em um biorreator à membrana (BRM) tratando esgoto sanitário. Tal avaliação foi conduzida em BRM construído em escala de bancada, com volume útil de 15 L, operado por 420 dias na modalidade de batelada sequencial. Durante o período experimental, foram aplicadas 3 estratégias operacionais, E-1, E-2 e E-3, em que foram testadas as idades de lodo de 80, 40 e 20 dias, respectivamente. Os resultados revelaram que a utilização da idade de lodo de 20 dias resultou em licor misto com maior potencial incrustante, apresentando, neste caso, uma velocidade de colmatação (VC) das membranas de 1,95 mbar dia-1, aproximadamente 2 vezes maior do que a observada nas idades de lodo de 80 e 40 dias. A maior colmatação observada foi atribuída a maior concentração de produtos microbianos solúveis (PMSs) no licor misto e a maior relação proteínas/polissacarídeos (PN/PS) dos flocos biológicos nesse período em questão. Por outro lado, a aplicação da idade de lodo de 80 dias resultou em menor VC das membranas do BRM, com valor de 0,82 mbar dia-1. Contudo, no período final dessa estratégia foi observado crescimento excessivo de bactérias filamentosas, que se refletiu em piora da filtrabilidade do licor misto e aumento da VC das membranas. De maneira geral, os resultados obtidos mostraram que a aplicação da idade de lodo de 40 dias resultou em licor misto com menor potencial incrustante.

ABSTRACT This study evaluated the effect of solids retention time (SRT) on membrane fouling rate in a membrane bioreactor (MBR) treating municipal wastewater. The evaluation was conducted in a membrane bioreactor built in bench scale, with a volume of 15 L, operated for 420 days in the sequential batch regime. During this period, three experimental runs were applied, E-1, E-2 and E-3, in which the solids retention time of 80, 40 and 20 days, respectively, were tested. The results showed that use of 20-days solids retention time resulted in a higher membrane fouling rate (MFR), with value of 1,95 mbar d-1, approximately two times higher than observed in the solids retention time of 80 and 40 days. The higher membrane fouling rate observed was attributed to a higher concentration of soluble microbial products (SMP) in the mixed liquor and to the higher proteins/polysaccharides ratio of the biological flocs in this period. On the other hand, the use of 80-days solids retention time resulted in a lower membrane fouling rate, with a value of 0.82 mbar d-1. However, it was observed in the final period of this experimental run an excessive growth of filamentous bacteria, which was reflected in a deterioration of the mixed liquor filterability and an increase of membrane fouling rate. Overall, the results showed that the 40-days solids retention time resulted in a mixed liquor with lower fouling propensity.

Produção de L-asparaginase pela levedura Leucosporidium muscorum CRM 1648 isolada de sedimento marinho coletado na Península Antártica / L-asparaginase production by the yeast Leucosporidium muscorum CRM 1648 isolated from marine sediments collected in Antarctic Peninsula

L-asparaginase (L-ASNase) é uma enzima com propriedades interessantes para a indústria médica, farmacêutica e de alimentos, que tem recebido atenção especial, inclusive no Brasil, por fazer parte do protocolo de tratamento de distúrbios linfoproliferativos, como a leucemia linfoblástica aguda (LLA). No mercado desde a década de 1970, as enzimas de origem bacteriana enfrentam algumas limitações por provocarem reações adversas graves em quase 80% dos pacientes em tratamento. Nesse contexto, L-ASNases provenientes de leveduras se destacam como alternativa, por serem mais próximas às congêneres humanas. A Antártica ainda é um ambiente pouco explorado, com grande diversidade de microrganismos com potencial para a produção de moléculas biológicas de interesse industrial. Nesse contexto, 150 leveduras isoladas de amostras de sedimento marinho coletadas na Península Antártica como parte do projeto MICROSFERA (PROANTAR/CNPq) foram avaliadas para a produção de L-ASNase. A triagem resultou em 9 isolados produtores, dos quais 7 pertencem ao gênero Leucosporidium. A linhagem L. muscorum CRM 1648 foi a que produziu mais enzima (540 U.L-1), com maior produtividade (5,6 U.L-1.h-1) e, por isso, foi alvo deste estudo. A análise univariada de fontes de carbono e nitrogênio indicou maior crescimento desse microrganismo e produção de L-ASNase em meio CD com extrato de levedura, prolina e sacarose. Ureia, cloreto de amônio e sulfato de amônio resultaram em baixa ou nenhuma produção da enzima, sugerindo que a metabolização de fontes de nitrogênio por essa linhagem está sob a influência do fenômeno de repressão catabólica pelo nitrogênio (RCN). Dois delineamentos experimentais do tipo fatorial completo resultaram em um aumento de 10 vezes na produção e produtividade da enzima (4582,5 U.L-1 e 63,6 U.L-1.h-1, respectivamente). A análise univariada da concentração inicial de inóculo (X0), pH inicial do meio, temperatura e adição de água do mar mostrou que a melhor condição para a produção foi: pH = 5,5 ou 6,5, cultivo a 15°C com adição de água do mar (25-50% m/v). A variável X0 não foi significativa nas concentrações avaliadas. Cultivos em biorreator (batelada) foram conduzidos em quatro diferentes níveis de oxigênio dissolvido (OD): (1) OD não controlado e abaixo de 20%, (2) OD não controlado e acima de 20%, (3) OD controlado em 80% e (4) OD controlado em 20%. Os resultados mostraram que OD é fator limitante para o crescimento de L. muscorum CRM 1648 e produção de L-ASNase por essa levedura e deve ser mantido acima de 35% para maior produção da enzima.Neste trabalho, a composição do meio e condições de cultivo foram estabelecidas para favorecer a produção de uma nova L-ASNase livre de atividade glutaminásica por levedura adaptada ao frio, abrindo espaço para novos estudos acerca de seu potencial antileucêmico e possível uso como alternativa às enzimas já existentes no mercado no tratamento de LLA

L-asparaginase (L-ASNase) is an enzyme with interesting properties for medical, pharmaceutical and food industry, which has received special consideration, especially in Brazil, for being part of lymphoproliferative disorders treatment, such as acute lymphoblastic leukemia (ALL). Bacterial enzymes are on the market since the 1970s and face some limitations related to theirserious adverse reactions that reach almost 80% of all patients in treatment. In this context, L-ASNases from yeasts are highlighted as important alternative to bacterial enzymes, due to the closerphylogeny to human congeners. Antarctic environment has much to be explored, with a vast diversity of microorganisms with potential to produce biomolecules with industrial interest. A total of 150 yeasts isolated from Antarctic marine sediments as part of MICROSFERA project (PROANTAR/CNPq) were evaluated for L-ASNase production. The screening resulted in 9 producers, 7 species from the genus Leucosporidium. L. muscorum CRM 1648 was the strain that yielded the highest L-ASNase activity (540 U.L-1) and volumetric productivity (5.6 U.L-1.h-1). Carbon and Nitrogen sources were evaluated by a method of one-factor at a time (OFAT). From the gather results, sucrose, yeast extract and proline resulted in a maximal growth and highest enzyme production.The absence or low production of L-ASNase in medium with urea, ammonium chloride and ammonium sulfate suggests the presence of nitrogen catabolic repression (NCR). Carbon and nitrogen concentration were evaluated by full factorial design and yielded about ten times higher enzyme and volumetric productivity (4582.5 U.L-1 and 63.6 U.L-1.h-1, respectively). Initial inoculum concentration (X0), initial pH, temperature and concentration of seawater in the culture were evaluated by OFAT analysis and the best condition for L-ASNase production was: pH = 5.5 or 6.5, at 15 °C with addition of seawater (25-50 wt%). X0 was not considered a significant variable. Bioreactor assays (in batch regime) were performed in four different dissolved oxygen (DO) levels: (1) without DO control (DO remained under 20%), (2) without DO control (DO remained above 20%), (3) DO controlled at 80%, and (4) DO controlled at 20%.The results showed that DO is a key factor for growth of L. muscorum CRM 1648 and production of L-ASNase by this yeast and should be maintained above 35% for higher production of this enzyme.At this work, the medium and culture conditions were established to support the production of a novel glutaminase-free L-ASNase by a cold adapted yeast, opening a new path for further studies regarding its antileukemic potential and possible use as an alternative for ALL treatment

Advances in multi-scale analysis and regulation for fermentation process / 生物工程学报

Industrial fermentation focuses on realizing the uniform of high titer, high yield, and high productivity. Multi-scale analysis and regulation, including molecule level, cell level, and bioreactor level, facilitate global optimization and dynamic balance of fermentation process, which determine high efficiency of biosynthesis, targeted directionality of bioconversion, process robustness, and well-organized system. In this review, we summariz and discuss advances in multi-scale analysis and regulation for fermentation process focusing on the following four aspects: 1) kinetic modeling of metabolic pathways, 2) characteristic of cell metabolism, 3) co-coupling fermentation and purification, and 4) bioreactor design. Integrating multi-scale analysis of fermentation process and integrating multi-scale regulation are expected as an important strategy for realizing highly efficient fermentation by industrial microorganisms.

Current Progress in Tendon and Ligament Tissue Engineering

BACKGROUND: Tendon and ligament injuries accounted for 30% of all musculoskeletal consultations with 4 million new incidences worldwide each year and thus imposed a significant burden to the society and the economy. Damaged tendon and ligament can severely affect the normal body movement and might lead to many complications if not treated promptly and adequately. Current conventional treatment through surgical repair and tissue graft are ineffective with a high rate of recurrence.METHODS: In this review, we first discussed the anatomy, physiology and pathophysiology of tendon and ligament injuries and its current treatment. Secondly, we explored the current role of tendon and ligament tissue engineering, describing its recent advances. After that, we also described stem cell and cell secreted product approaches in tendon and ligament injuries. Lastly, we examined the role of the bioreactor and mechanical loading in in vitro maturation of engineered tendon and ligament.RESULTS: Tissue engineering offers various alternative ways of treatment from biological tissue constructs to stem cell therapy and cell secreted products. Bioreactor with mechanical stimulation is instrumental in preparing mature engineered tendon and ligament substitutes in vitro.CONCLUSIONS: Tissue engineering showed great promise in replacing the damaged tendon and ligament. However, more study is needed to develop ideal engineered tendon and ligament.