Immobilizing engineered Escherichia coli cells into zeolitic imidazolate framework 8 for efficient biosynthesis of Ala-Gln / 生物工程学报

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.

Effects of glutamine and alanine supplementation, in their free form or as dipeptide, on fatigue parameters of rats submitted to resistance training / Efeitos da suplementação com glutamina e alanina, na forma livre ou como dipeptídeo, sobre parâmetros de fadiga de ratos submetidos ao treinamento resistido

Fatigue is defined as the inability to maintain muscle power and strength, impairing performance. Nutritional interventions have been used to delay this phenomenon, such as glutamine and alanine supplementation. These amino acids might attenuate several causes of fatigue, since they are important energy substrates, transport ammonia avoiding the accumulation of this toxic metabolite and attenuate muscle damage and oxidative stress. Thus, the aim of this study was to evaluate the effects of glutamine and alanine supplementation on central and muscle fatigue parameters of rats submitted to resistance training (RT). Forty adult Wistar rats (60 days) were distributed into five groups: SED (sedentary, receiving water), CON (trained, receiving water), ALA, G+A and DIP (trained and supplemented with alanine, glutamine and alanine in their free form, and Lalanyl-L-glutamine, respectively). Trained groups underwent a ladder-climbing exercise, with progressive loads, for eight weeks. Supplements were diluted in water to a 4% concentration and offered ad libitum during the last 21 days of experiment. RT increased plasma glucose, the muscle concentrations of ammonia and glutathione (GSH) and the muscle damage parameters - plasma creatine kinase (CK) and lactate dehydrogenase (LDH), whereas decreased muscle glycogen. G+A supplementation prevented the increase of muscle ammonia by RT, while ALA and G+A administration reduced plasma CK and LDH, and DIP supplementation increased the muscle content of glycogen and LDH. Contrary to expectations, DIP administration increased central fatigue parameters, such as plasma concentration of free fatty acids (FFA), hypothalamic content of serotonin and serotonin/dopamine ratio. Despite these results, there was no difference between groups in the maximum carrying capacity (MCC) tests. In conclusion, supplementation with glutamine and alanine improves some fatigue parameters, but does not affect physical performance of rats submitted to RT

O termo fadiga é definido como a incapacidade de manutenção da força e da potência musculares, prejudicando a performance. Intervenções nutricionais têm sido utilizadas para retardar este fenômeno, como a suplementação com glutamina e alanina. Estes aminoácidos poderiam atenuar diversas causas de fadiga, pois são importantes substratos energéticos, carreiam amônia evitando o acúmulo deste metabólito tóxico e atenuam a lesão muscular e o estresse oxidativo. Logo, o objetivo deste estudo foi avaliar os efeitos da suplementação com glutamina e alanina sobre parâmetros de fadiga central e muscular em ratos submetidos ao treinamento resistido (TR). Foram utilizados 40 ratos Wistar adultos (60 dias de idade), distribuídos nos grupos: SED (não treinados, recebendo água), CON (treinados, recebendo água), ALA, G+A e DIP (treinados e suplementados com alanina, glutamina e alanina livres, e L-alanil-L-glutamina, respectivamente). Os grupos treinados realizaram um exercício de escalada em escada, com aumento progressivo de carga, durante oito semanas. A suplementação foi diluída a 4% em água e ofertada via oral, ad libitum, durante os últimos 21 dias de experimento. O TR aumentou a glicemia, as concentrações musculares de amônia e de glutationa (GSH) e os parâmetros de lesão muscular - creatina quinase (CK) e lactato desidrogenase (LDH) no plasma, enquanto reduziu o glicogênio no músculo. A suplementação com G+A preveniu o aumento de amônia muscular promovido pelo TR, enquanto a administração de ALA e G+A reduziu as concentrações de CK e LDH no plasma, e a suplementação com DIP aumentou o conteúdo muscular de glicogênio e de LDH. Ao contrário do esperado, a administração de DIP aumentou parâmetros de fadiga central, como as concentrações plasmáticas de ácidos graxos livres, o conteúdo hipotalâmico de serotonina e a razão serotonina/dopamina. Apesar disso, não houve diferença entre os grupos nos testes de carga máxima. Em conclusão, a suplementação com glutamina e alanina melhora alguns parâmetros de fadiga, mas não afeta o desempenho físico em ratos submetidos ao TR

Expression, purification, characterization and application of α-amino acid ester acyltransferase from recombinant Escherichia coli / 生物工程学报

α-Amino acid ester acyltransferase (Aet) catalyzes the L-alanyl-L-glutamine forming reaction from L-alaine methylester hydrochloride and L-glutamine. In this study, the recombinant Escherichia coli saet-QC01 was used to express the α-amino acid acyltransferase, and its expression conditions were optimized. The recombinant protein was separated and purified by Ni-NTA affinity chromatography, and its enzymatic properties and catalytic applications were studied. The induction conditions suitable for enzyme production optimized were as follows: The temperature was 20 ℃, the induction stage (OD₆₀₀=2.0-2.5), IPTG concentration was 0.6 mmol/L, induction time was 12 h. The optimal reaction conditions of α-amino acid acyltransferase were 27 ℃, pH 8.5, it was most stable between pH 7.0 and 8.0 and relatively stable in an acidic environment, and low concentration of Co²⁺ or EDTA could promote the enzyme activity. Under optimal reaction conditions, 600 mmol/L of L-alaine methylester hydrochloride and 480 mmol/L of L-glutamine, the yield of L-alanyl-L-glutamine reached 78.2 g/L and productivity of 1.955 g/L/min, the conversion rate reached 75.0%. α-Amino acid ester acyltransferase has excellent acid-basei resistance, high catalytic efficiency. These characteristics suggest its application prospects in the industrial production.

Efeito da suplementação com L-glutamina e L-alanina, livres ou como dipeptídeo, sobre a lesão, inflamação e citoproteção em modelos de estresse in vivo e in vitro / Effects of supplementation with L-glutamine and L-alanine, in their free form or as dipeptide, on muscle damage, inflammation and cytoprotection of in vivo and in vitro stress models

Subprojeto 1: Determinação do efeito anti-inflamatório e citoprotetor da suplementação com L-glutamina e L-alanina, ou com L-alanil-L-glutamina (DIP) em ratos submetidos a treinamento resistido. Exercícios intensos reduzem a disponibilidade de glutamina, comprometendo a função imune e a recuperação de atletas. O objetivo do estudo foi avaliar os efeitos da suplementação oral crônica com L-glutamina e L-alanina, nas formas livres ou como dipeptídeo (DIP), sobre parâmetros de lesão, inflamação e citoproteção em ratos Wistar adultos submetidos a treinamento resistido (TR). Neste estudo, o TR reduziu a concentração de glutamina no plasma e no músculo EDL. No entanto, este efeito foi atenuado pelos suplementos contendo L-glutamina, os quais aumentaram os conteúdos da proteína de resposta ao estresse (HSP70) em células do sistema imune (PBMC) e no EDL, concomitantemente à redução da ativação do NF-kB e a da concentração de citocinas no EDL. O efeito protetor das suplementações também foi evidenciado pela atenuação de marcadores de lesão (CK e LDH) e inflamação (TNF-α e IL-1ß), bem como pelo aumento nas concentrações de marcadores anti-inflamatórios (IL-6, IL-10 e MCP-1) no plasma. Nossos resultados sugerem que a suplementação oral crônica com L-glutamina (administrada com L-alanina livre ou como DIP) promoveu efeitos citoprotetores mediados pela HSP70 em resposta à lesão e inflamação induzidas pelo TR. Subprojeto 2: Efeitos da L-alanil-L-glutamina sobre as vias de sinalização da insulina e da mTOR/S6K, e citoproteção em células musculoesqueléticas C2C12. O dipeptídeo L-alanil-L-glutamina é conhecido por modular o metabolismo e a viabilidade celular. Contudo, os efeitos sobre os componentes clássicos das vias de sinalização da insulina e da mTOR/S6K, bem como o efeito citoprotetor em células musculares, são pouco esclarecidos. O objetivo deste estudo foi investigar o efeito do DIP sobre as vias de sinalização da insulina e da mTOR/S6K em miotubos C2C12, em condições normais ou resistentes à insulina. A exposição crônica à insulina (24h) promoveu resistência à insulina, reduzindo os conteúdos totais do receptor beta (IR-ß) e do substrato do receptor de insulina (IRS-1), e diminuindo a fosforilação de IRS-1, AKT e P44/42 MAPK. Adicionalmente, houve redução na expressão do transportador de glicose (GLUT4) e HSP70, redução da viabilidade celular e menor fosforilação de p70S6k e S6, proteínas relacionadas à síntese proteica. Em contraste, a suplementação com DIP aumentou os conteúdos totais de IR-ß e IRS-1 e a fosforilação de IRS-1 e AKT. A glicólise anaeróbia e a capacidade glicolítica, além da fosforilação de p70S6k e S6, foram aumentadas pelo DIP em condições normais e na resistência à insulina. Nestas condições experimentais, nossos resultados sugerem que a suplementação com DIP melhorou as vias de sinalizações da insulina e da mTOR/S6K, aumentou a captação e metabolização da glicose, independente da estimulação com insulina e, finalmente, promoveu citoproteção resgatando parcialmente as células de um estado resistente à insulina, por meio do aumento de HSP70 e ativação das etapas finais da via mTOR/S6K.

Subproject 1: Determination of the anti-inflammatory and cytoprotective effects of supplementation with L-glutamine and L-alanine, or with L-alanyl-L-glutamine in rats submitted to resistance training. Intense exercise reduces glutamine availability, compromising immune function and recovery of athletes. The objective of the study was to evaluate the effects of chronic oral supplementation with L-glutamine and L-alanine, in their free form or as dipeptide (DIP), on muscle damage, inflammation and cytoprotection in adult Wistar rats submitted to resistance training (RT). In this study, RT reduced glutamine concentration in plasma and EDL muscle. However, this effect was attenuated by supplements containing L-glutamine, which increased the contents of the stress response protein (HSP70) in immune system cells (PBMC) and EDL, concomitantly with the reduction of NF-kB activation and the concentration of cytokines in EDL. The protective effect of supplementation was also evidenced by attenuation of lesion markers (CK and LDH) and inflammation (TNF-α and IL-1ß), as well as by the increase in anti-inflammatory plasma markers (IL-6, IL-10 and MCP-1). Our results suggest that chronic oral supplementation with L-glutamine (administered along with free L-alanine or as DIP) promoted HSP70-mediated cytoprotective effects in response to RT-induced injury and inflammation. Subproject 2: Effects of L-alanyl-L-glutamine on the components of insulin and mTOR/ S6K signaling pathways and cytoprotection in C2C12 musculoskeletal cells. The dipeptide L-alanyl-L-glutamine is known to modulate metabolism and cell viability. However, the effects on the classical components of insulin and mTOR/ S6K signaling pathways, as well as the cytoprotective effect on muscle cells, are poorly understood. The aim of this study was to investigate the effect of DIP on insulin and mTOR/ S6K signaling pathways in C2C12 myotubes, under normal or insulin resistant conditions. Chronic insulin exposure (24h) promoted insulin resistance, reducing the total contents of the insulin receptor (IR-ß) and the insulin receptor substrate (IRS-1), and decreasing the phosphorylation of IRS-1, AKT and P44/ 42 MAPK. In addition, there was a reduction in the expression of glucose transporter (GLUT4) and HSP70, reduction of cell viability and defective phosphorylation of p70S6k and S6, which are related to protein synthesis. On the other hand, DIP supplementation increased the total contents of IR-ß and IRS-1 and the phosphorylation of IRS-1 and AKT. Anaerobic glycolysis and glycolytic capacity, in addition to phosphorylation of p70S6k and S6, were increased by DIP under normal conditions and in insulin resistance. In our experimental conditions, our results suggest that DIP supplementation improved the signaling pathways of insulin and mTOR/ S6K, increased glucose uptake and metabolism, independent of insulin stimulation, and finally promoted cytoprotection by partially rescuing the cells of an insulin resistant state, by increasing HSP70 and activating the final stages of the mTOR/ S6K pathway.