Advances on microbial synthesis of L-proline and trans-4-hydroxy-L-proline / 生物工程学报

L-proline (L-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-L-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of L-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of "green, environmental protection and high efficiency". In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of L-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of L-Pro and T-4-Hyp. This paper reviews the application and production methods of L-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of L-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of L-Pro and T-4-Hyp, aiming to provide a theoretical basis for the "green bio-manufacturing" of L-Pro and T-4-Hyp and promote their industrial production.

Multivariate optimization of Staphylococcus xylosus AD1biomass production using sugarcane molasses plus yeast extract and soybean meal

Staphylococcus xylosusis a microorganism that has important physiological and technological characteristics that make it suitable for use as a starter culture in fermented meat products. For the development of these products in the food industry, it is necessary to produce biomass by the multiplication of starter cultures using low-cost media. This study developed a culture medium based on sugarcane molasses (SCM) supplemented with yeast extract (YE) and soybean meal (SM) to produce S. xylosusAD1 biomass employing a Box Behnken multivariateoptimization design,usingthe best concentrations of the constituents of the culture medium for S. xylosusAD1 growth. By combining the mathematical models by the desirability function, it was possible to establish the optimal condition for the maximum production of viable cells and biomass. The optimal experimental condition was found when the fermentative process medium was composed of 10% SCM, 2% YE and 4% SM. In addition, the results of all experiments, except for the medium formulated with only SCM,presented a better performance than the commercial medium Brain Heart Infusion for the growth of S. xylosusAD1. The culture medium with agro-industrial byproduct (SCM) supplemented with YE and SM is an excellent alternative for producing S. xylosusAD1 biomass.

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.

Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.

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.

Effect of fermentation conditions on the production of hyaluronic acid by Streptococcus zooepidemicus ATCC 39920 / Efeito das condições de fermentação na produção de ácido hialurônico por Streptococcus zooepidemicus ATCC 39920

The production of hyaluronic acid by Streptococcus zooepidemicus ATCC 39920 with varying rates of pH (6.0, 7.0, 8.0), temperature (34; 37; 40°C), agitation (100, 150, 200 rpm), glucose (10, 20, 30 g L -1) and yeast extract concentration (10, 20, 30 g L -1) was evaluated by statistical approaches. The best conditions for the production of hyaluronic acid was pH 8.0, 37°C and 100 rpm in a medium containing 30 g L- 1 glucose and yeast extract, for a production of 0.787 g L- 1. Temperature, pH and yeast extract were significant variables (p < 0.05). Yeast extract and pH had a positive effect on the production of the polymer. Lactate, formate and acetate synthesis were also analyzed. Current assay showed the feasibility of statistical tools to optimize the physical and nutritional parameters for the production of hyaluronic acid and the improvement of the fermentation process.

A produção de ácido hialurônico por Streptococcus zooepidemicus ATCC 39920 foi avaliada variando pH (6,0; 7,0, 8,0), temperatura (34; 37; 40°C), agitação (100, 150, 200 rpm) e concentração de glicose (10, 20, 30 g L-1) e extrato de levedura (10, 20, 30 g L-1) por metodologias estatísticas. A condição otimizada foi pH 8,0, 37°C e 100 rpm, em meio contendo 30 g L-1 de glicose e extrato de levedura atingindo a produção de 0,787 g L-1. O pH, temperatura e extrato de levedura foram as variáveis significativas (p < 0,05). Extrato de levedura e pH apresentaram efeito positivo para a produção do polímero. A síntese de ácido lático, fórmico e acético também foi analisada. Este estudo demonstra a viabilidade de utilização de ferramentas estatísticas para otimizar os parâmetros físicos e nutricionais para a produção de ácido hialurônico, permitindo a melhoria do processo fermentativo.

Parâmetros digestivos e produção de proteína microbiana em novilhas em crescimento compensatório / Digestive parameters and protein microbial production in heifers under compensatory growth

Avaliaram-se o manejo para crescimento compensatório e o efeito da suplementação com ionóforo na dieta sobre os parâmetros digestivos e sobre a produção de proteína microbiana de novilhas leiteiras. Foram utilizadas 20 animais puros da raça Pardo-Suíça, com média de peso inicial de 200kg aos cinco meses de idade. Os tratamentos foram arranjados em um esquema fatorial 2x2x2, e os animais foram alocados, aleatoriamente, em cada uma das combinações. O fator 1 consistiu dos sistemas de alimentação (convencional e crescimento compensatório), o fator 2, da utilização (200mg de monensina/animal/dia) ou não de ionóforo e o fator 3, dos períodos de alimentação (P1 e P2). A inclusão de ionóforo na dieta aumentou os coeficientes de digestibilidade total da matéria seca, da matéria orgânica, dos carboidratos totais e da fibra em detergente neutro. Não houve efeito do sistema de alimentação, da adição de ionóforo à dieta e do período sobre a produção microbiana. A eficiência microbiana (g PB microbiana/kg de NDT consumido) no período de restrição foi maior que no período de realimentação.

The effects of compensatory growth and ionophore supplementation of diet of dairy heifer on digestive parameters and protein microbial production were evaluated. Twenty five-month-old Brown-Swiss heifers averaging 200kg b.w. were used. The treatments were arranged in a factorial design (2x2x2) with the animals randomly allocated to each of the combinations. Factor 1 was based on the feeding systems (conventional and compensatory growth), factor 2 on ionophore supplementation option (200mg of monensin/animal/day or not) and factor 3, on the feeding periods (P1 and P2). The diet supplemented with ionophore increased the total digestibility coefficients of dry matter, organic matter, total carbohydrates, and neutral detergent fiber. No effect of feeding systems, ionophore supplementation, or feeding periods based on microbial production was oberved. The microbial efficiency (g of microbial crude protein/kg of NDT intake) during the restriction period was higher than the re-feeding period.