Metabolic Mechanism And Applications Of The Exopolysaccharide From Bacillus Licheniformis

Extracellular polymeric substances are macromolecular polymers secreted by microorganisms.Due to diverse advantages such as biodegradability,high efficiency,non-toxicity and non-secondary pollution,extracellular polymeric substances have the potential to replace common chemical synthetic polymers.The applications of extracellular polymeric substances,partiticularly with regard to bioflocculants,have attracted much attention in recent years.In this paper,the synthetic process,fermentation production and practical applications of bacterial extracellular polymers were systematically studied.All results are as following:1)The present study reports the sequenced genome of Bacillus licheniformis CGMCC 2876,which is composed of a 4,284,461 bp chromosome that contains 4,188 protein-coding genes,72 tRNA genes,and 21 rRNA genes.16 genes related to polysaccharide production were identified in the B.licheniformis genome.These genes are distributed in different locations across the genome and encode proteins responsible for the conversion of glucose into the units that compose polysaccharides.Additional analysis revealed an eps gene cluster with 16 open reading frames.Conserved Domains Database analysis combined with qPCR experiments indicated that all genes in this cluster were involved in polysaccharide synthesis.Phosphoglucomutase and UDP-glucose pyrophosphorylase were supposed to be key enzymes in polysaccharide secretion in B.licheniformis.A biosynthesis pathway for the production of exopolysaccharide involving the integration of individual genes was proposed based on functional analysis.Overexpression of the UDP-glucose pyrophosphorylase gene gtaB-2 not only increased the flocculating activity by 71%but also increased bioflocculant yield by 13.3%.Independent of UDP-N-acetyl-D-mannosamine dehydrogenase gene wbpA-2,flocculating activity and polysaccharide yield were negatively impacted by overexpression of the UDP-N-acetylglucosamine 2-epimerase gene mnaA.Overexpression of epsDEF from the eps gene cluster in B.licheniformis CGMCC 2876 increased the flocculating activity of the recombinant strain by 90%compared to the original strain.Similarly,the crude yield of polysaccharide bioflocculant was enhanced by 27.8%.Overall,epsDEF and gtaB2 were identified as key genes for exopolysaccharide synthesis in B.licheniformis.These results will be useful for further engineering of B.licheniformis for industrial bioflocculant production.2)The present study determined that ?-glucosidase played a negative role in bioflocculant synthesis.The gene encoding ?-glucosidase was cloned and expressed in Escherichia coli BL21.This gene consists of 1437 bp and encodes 478 amino acid residues.The recombinant ?-glucosidase(Bgl.blil)was purified and showed a molecular mass of 53.4 kDa by SDS-PAGE.The expression conditions of Bgl.blil were optimized;the activity of ?-glucosidase reached a maximum of 45.44 U mL-1.Glucose clearly inhibited the activity of P-glucosidase.The purified recombinant Bgl.blil hydrolysed polysaccharide bioflocculant in vitro and synergised with other cellulases.The ability of Bgl.blil to hydrolyse polysaccharide bioflocculant was the reason for the decrease in flocculating activity and indicated the utility of this enzyme for diverse industrial processes.3)Bacillus licheniformis-epsDEF?Alteromonas sp.CGMCC 10612 and Pseudomonas fluorescens mutant T4-2 were cultivated in a volume of 2 L for scaling up and fermentation processes of extracellular polymers were studied.Production of the extracellular polymers of these three strains was positively associated with cell growth.Compared with the shake flask fermentation,the output of the extracellular polymers and the flocculation activity were improved.The crude yield of extracellular polymers from B.licheniformis-epsDEF?Alteromonas sp.CGMCC 10612 and P.fluorescens mutant T4-2 was 11.77,11.18 and 6.36 g L-1,repectively.The applications of these three kinds of extracellular polymers were studied.MBF-B from B.licheniformis-epsDEF was applied to microalgae collection.The optimum dosage of MBF-B was 80 mg L-1 and the flocculating efficiency of microalgae was as high as 80%in a wide range of pH.MBF-A from Alteromonas sp.CGMCC 10612 was applied to colour removal from the wastewater.The results showed MBF-A was able to remove Congo Red,Direct Black and Methylene Blue at efficiencies of 98.5%,97.9%and 72.3%respectively.MBF-P from P.fluorescens mutant T4-2 was applied to metal removal.The adsorption capacity of Cr(?)by MBF-P reached as high as 80.13 mg g-1 with a 55 min-equilibrium time in acidic conditions(pH=2.0).As for As(?),the adsorption capacity was 45.93 mg g-1 with a 35 min-equilibrium time in neutrallty condition(pH=7.0).These results can provide basic data and theoretical basis for the industrial application and development of extracellular polymers.