The Physiological Metabolism Of The Curdlan-producing Alcaligenes Faecalis

Curdlan is a new kind of microbial exopolysaccharide synthesized by Alcaligenes faecalis or Agrobacterium sp. The unique rheological and thermal gelling properties of curdlan can be applied advantageously to the fields of food, pharmaceutical and architecture. Curdlan was synthesized under nitrogen-limited condition, the synthesis and activity of intracellular enzyme was greatly restrained at the condition, leading to the decreasing productivity of curdlan and the hard control of fermentation process. In this study, we first investigated the total protein expression change of Alcaligenes faecalis when the condition changed from nitrogen-sufficient to nitrogen-limited by two-dimensional electrophoresis (2-DE). Then the change of physiological metabolism between Alcaligenes faecalis and its ntrC mutant was compared. The results helped us to understand the regulation mechanism of nitrogen on the curdlan synthesis. Finally, the effect of Mn2+ concentration on curdlan production was observed.A method for high resolution and good repeatability 2-DE protein map construction was firstly developed. A lysis buffer was used for extracting total cellular protein, and linear pH 4-7 immobilized pH gradients (IPG) gel strips were used for the first dimension electrophoresis. Compared by two staining methods (coomassie blue staining and silver staining), it was found that silver staining could get more clearly protein spots in 2-DE map.The overall protein expression of Alcaligenes faecalis under nitrogen-sufficient and nitrogen-limited conditions was examined and the expression levels of 20 proteins were significantly changed. Three differential proteins were successfully identified by MALDI-TOF-MS, and the enoyl-(acyl carrier protein) reductase is associated with the stunted cell growth.We also compared the bacterial differentiation rate and the capacity of curdlan production between Alcaligenes faecalis and its ntrC mutant. The differentiation rate of ntrC mutant was obviously quickly than the wild type after cultivated 18 hours and the yield of curdlan was significantly decreased which proves that the gene ntrC is a global regulator for regulating curdlan synthesis.The proteins expression difference between Alcaligenes faecalis and its ntrC mutant was examined by 2-DE.40 proteins were not expressed in the ntrC mutant and the levels of other 43 proteins were markedly changed. Four of them were identified by MS; the N-acetyl-gamma-glutamyl-phosphate reductase related to the metabolism of nitrogen and the nucleoside diphosphate kinase connected with the curdlan synthesis.At last, the effect of Mn2+ concentration on production of curdlan was investigated. We found that the productivity of curdlan reached to 22.3 g/L and 45.3% higher than the control when MnCl2 concentration increased to 0.13 g/L at 18 hours in flask fermentation. And the concentration of intracellular UDP-Glucose decreased after Mn2+ addition. The results suggest that Mn2+ may stimulate the key enzyme glucosyltransferase about curdlan synthesis.