The Analysis Of Key Factors In Exopolysaccharide Synthesis By Bacillus Mucilaginosus And The Metabolic Regulation

Bacillus mucilaginosus SM-01 preserved in our lab could produce an exopolysaccharide named BMPS（Bacillus mucilaginosus polysaccharide）. BMPS had good physicochemical properties which had wide applications in surgical dressing, pharmaceutical carriers and immunological enhancement. In this work, the effects of nitrogen source concentration and calcium salts on BMPS biosynthesis were studied. The physiological and metabolic changes under nitrogen-limited cultivation and CaCO₃ addition based on comparative proteomics technology, and the metabolic regulation strategy to enhance the BMPS productivity by B. mucilaginosus SM-01 were systematically studied. The main results are as follows:（1） The two-dimensional electrophoresis（2-DE） technology system of total proteins of B. mucilaginosus SM-01 was established by the following optimized conditions: lower hydrochloric acid solution to dissolute expolysaccharide; TCA/acetone combined with phenol extraction to extract total proteins; 17 cm linear IPG strip with pH4-7 and program Ⅱ to isoelectric focusing. Under the optimized conditions, the 2-DE maps with clear background and high resolution were obtained.（2） Effects of nitrogen-limited cultivation on BMPS production and proteomics analysis were studied. Under nitrogen-limited cultivation（urea 0.3 g/L,glocuse 60 g/L,C/N=171.8）, the dry cell weight（DCW） of B. mucilaginosus SM-01 and BMPS concentration reached 2.6 g/L and 26.1 g/L, which was 40.6% and 1.9-fold of that under nitrogen-replete cultivation（urea 3.0 g/L,glocuse 60 g/L,C/N=17.6）. Based on 2-DE and mass spectrometry technology, 34 proteins were successfully identified, of which 25 proteins were down-regulated and 9 proteins were up-regulated under nitrogen-limited cultivation. The KEGG pathway analysis revealed that enzymes concerning carbohydrate metabolism, amino acid biosynthesis and nucleotide metabolism were depressed. Whereas mannosyltransferase involved in polysaccharide biosynthesis was overexpressed.（3） Effects of CaCO₃ on BMPS production and proteomics analysis were studied. With 5 g/L CaCO₃ addition, the DCW, BMPS concentration and substrate utilization were 2.6-fold, 11.2-fold and 3.3-fold higher than that without CaCO₃ addition. The specific rate of growth and BMPS synthesis were also improved. Based on 2-DE and mass spectrometry technology, 34 proteins were successfully identified, of which 13 up-regulated proteins and 21 down-regulated proteins with CaCO₃ addition. The KEGG pathway analysis revealed that enzymes concerning carbohydrate metabolism, amino acid biosynthesis and nucleotide metabolism were depressed. Whereas UDP-glucose pyrophosphorylase and proteins involved in energy metabolism were overexpressed.（4） Based on above researches, the metabolic regulation for enhancing the BMPS productivity by B. mucilaginosus SM-01 was studied. Mn²⁺ was added into the fermentation medium as a specific activator for glycosyltransferases. The results showed that the BMPS concentration reached 36.2 g/L with 0.15 g/L Mn Cl2·4H2O addition at 24 h in 5 L fermenter, which was 27.8% higher than control groups.