[Hearing screening for 2061 neonates and high risk factors for hearing disorders].
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Female↗
Biomedical subjects
Li-Ming Liu
Publications and source records attributed to Li-Ming Liu.
[Torulopsis glabrata neomycin-resistant mutant abolishes pyruvate production with enhancement of glucose consumption rate].
To further increase the rate of glucose consumption by multi-vitamin auxotrophic yeast Torulopsis glabrata. A neomycin-resistant mutant N07, with the activity of F1-ATPase decreased roughly 35% but glucose consumed per cell was increased 38% than that of parent strain, was breed based on analysis of energy metabolic pathway. The typical inhibitors of F1F0-ATPase, DCCD, NaN3 and neomycin, depressed the F1-ATPase activity of parental strain but no effect on that of mutant strain. Strain N07 was cultured in a pyruvate fermentation medium containing 100g/L of glucose using flask. It was found that the rate of glucose consumption and pyruvate production were higher by 34% and 42.9% in the mutant than in the parent, respectively. However, the rate and yield of growth (about 24%) of the mutant was lower than that of the parent. The content of intracellular ATP of the mutant also decreased 23.7% than that of the parent. The activities of key enzymes in glycolytic pathway and electron transfer chain of the mutant and the parent were determined. Enzymatic analysis revealed that, compared with the parent strain CCTCC M202019. The activities of key enzymes, phosphofructokinase, pyruvate kinase, glyceraldyde-3-phosphate dehydrogenase of the mutant N07 increased 63.7%, 28.8% and 14.4%, respectively, all the key enzymes of electron transfer chain in the mutant N07 also increased roughly 10%.
Adenosine Triphosphate↗
[Addition of TCA cycle intermediates enhances pyruvate production].
The capability of utilizing the intermediates of TCA-cycle as the sole carbon source by the multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019 under the conditions of vitamins limitation was demonstrated. Furthermore, the colony numbers grown on medium supplemented with glucose, acetate and one of the intermediates of TCA-cycle was higher than that of medium used glucose and acetate or medium used one of the intermediates of TCA-cycle carbon source. Among the intermediates of TCA-cycle used in this study, oxaloacetate was the best carbon source for the yeast and it was found that its presence stimulated the conversion of acetate to acetyl-CoA. In batch fermentation with glucose medium, the addition of 10 g/L of oxaloacetate improved the dry cell weight from 11.8 g/L to 13.6 g/L, and the productivity of pyruvate from 0.96 g x L(-1) x h(-1) to 1.19 g x L(-1) x h(-1), a 24% increase after 56 h growth. The yield of pyruvate on glucose was also improved as well, from 0.63 g/g to 0.66 g/g.
Candida glabrata↗
[The decrease of the activity of electron transfer chain of Torulopsis glabrata enhanced pyruvate productivity].
Torulopsis glabrata CCTCC M202019 was mutated by ethidium bromide to screen for respiratory-deficient mutants. Seven mutants that produced pyruvate higher than that of the parent were subjected to the tests of the capability assimilating fermentable substrate (glucose) and non-fermentable substrates (glycerol and acetate) to characterize true respiratory-deficient mutants. Mutants RD-16, RD-17 and RD-18 were unable to assimilate acetate or glycerol and were therefore identified as respiratory-deficient mutants. Compared to the parent strain, the growth the intracellular ATP content of those mutants decreased by 21% - 29% and 15% - 21%, respectively, while the glucose consumption per cell and the pyruvate production per cell of those mutants were enhanced by 20.7% - 30.7% and 30.7% - 55.5%, respectively. Qualitative analysis of cytochromes involved in electron transfer chain showed that mutants RD-16 and RD-18 lacked both cytochrome aa3 and b, while mutant RD-17 lacked cytochrome b. Enzymes analysis indicated that the activities of ATPase, succinate-cytochrome c reductase (complex I ), complex I + III , complex II + III, and complex IV of those mutants decreased by 14.6% - 22.2%, 34% - 41%, 38.6% - 52.6%, 21% - 25%, and 150% - 630%, respectively. However, increased glucose consumption per cell was not observed in those mutants, which might be due to that the NADH generated in glycolysis can not be completely oxidized via electron transfer chain. To avoid the accumulation of NADH, 2.1 mmol/L acetaldehyde was added to the culture broth of mutant RD-17 at 26h of fermentation. Using this strategy, the amount of pyruvate produced increased by 21.6% while the fermentation time was shortened from 62h to 48h.
Acetaldehyde↗
[CaCO3 stimulates alpha-ketoglutarate accumulation during pyruvate fermentation by Torulopsis glabrata].
A large amount of alpha-ketoglutarate (alpha-KG) (6.8 g/L) was accumulated in flask culture when CaCO3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. In a 5 L jar-fermentor, less alpha-KG (1.3 g/L) was produced when NaOH was used to adjust the pH, while more alpha-KG (11.5 g/L) detected when CaCO3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to alpha-KG (C(PYR)/ CalphaKG) was similar to that obtained in flask culture, suggesting the accumulation of alpha-ketoglutarate was related to the addition of CaCO3. Furthermore, it was found that: (1) delaying the addition time of CaCO3 decreased the a-ketoglutarate formation but increased C(PYR)/ C(alphaKG); and (2) under vitamin limitation conditions increasing the concentration of CaCO3 led to an increased a-KG accumulation at the expenses of pyruvate. To study which ions in CaCO3 was responsible for the accumulation of alpha-KG, the effects of different pH buffers on the a-KG accumulation were studied. The level of alpha-KG was found to correlate with the levels of both Ca2+ and CO3(2-), with Ca2+ played a dominant role and CO3(2-) played a minor role. To find out which pathway was responsible for the accumulation of alpha-KG, the effects of biotin and thiamine on alpha-KG accumulation was investigated. The increase in biotin concentration led to an increase in alpha-KG accumulation and a decrease in C(PYR)/ C(alpha-KG), while the levels of alpha-KG and C(PYR)/C(alphaKG) were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca2+ . On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca2+. To conclude, the higher level of a-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca2+, with CO3(2-) served as the substrate of the reaction.
Biotechnology↗
[Progress in biotechnological production of pyruvic acid].
Pyruvate, an important organic acid, is widely used in the industries of pharmaceuticals, chemicals, agrochemicals, food additives and so on. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost and high product quality. In this article, biosynthesis of pyruvate, including direct fermentative production and resting cell method as well as enzymatic method, was discussed. Furthermore, a comparison of these different methods was proposed. Since, a multi-vitamin auxotrophic strain of Torulopsis glabrata is the most competitive strain for industrial production of pyruvate, emphasis was therefore placed on the development of strains screening and fermentation optimization. Finally, some suggestions were put forward to improve the research in this field in the near future.
Biotechnology↗