Mutagenesis Of Acidiphilium Cryptum DX1-1 And Intracellular Proteins' Differential Expression Under Different Trophic Conditions

PHB is a kind of polyester stored in microorganism body as carbon source and energy source,when some prokaryote is in imbalance of carbon and nitrogen nourishment.As a kind of degradable microorganism material, besides the similar characteristics with chemosynthesis macromolecule,it also shows some specific characteristics that are not found in by ordinary chemosynthesis macromolecule,such as good optic activity,low oxygen filtering ability,anti-ultraviolet radiation,biological degradability,biological organism consistency,piezoelectricity,anti-cruor and so on.It has a promising future and has attracted more and more attention.Many companies and research institutes in the world carrying out the research and development work on degradable plastic.This summarize mainly introduces advances on the PHB study of the physical-chemical feature,inspecting method,biosynthesis,degradability,and genetic improvement of bacterium, and discusses its current application,existing problems,and possible resolving principles.A new bacterial strain,designated as strain DX1-1,was isolated from acid mine drainage of Dexing mine,Jiangxi Province,China.Furthermore, its capacity of producing polymer granules was identified by transmission electron microscope(TEM).The polymer granules was extracted and detected by infrared ray and ultraviolet radiation.The results showed that it had the same character with poly-β-hydroxybutyrate(PHB),which suggested that strain DX1-1 might have the potential of synthesizing biodegradable materials PHB.The strain Acidiphilium cryptum DX1-1 producing PHB was irradiated respectively by UV is and Co⁶⁰ to raise PHB production.The results indicated that the effect of UV better than using Co⁶⁰.One strain of the UV mutagenized called UV60-3 has the highest PHB production yield,showing final PHB concentration of 28.56 g/L,1.45 times higher than that of original strain.FT-IR spectroscopy analysis shows that the polymers obtained from the strain DX1-1 have the same IR spectra of standard PHB.Further research about the best appropriate C/N ratio of the mutant was done.The optimum ratio of C/N was approximately 0.94,the final PHB concentration reaches to 30.57g/L.Acidiphilium cryptum DX1-1 and UV60-3 were used in our comparative proteomic research,Intracellular proteins were extracted from UV60-3 cultivated with 30g/L(NH₄)₂SO₄ concentration,under the best situation for PHB production.After times of adjust parameter of two-dimensional electrophoresis(2-DE),finally got the appropriate condition.The protein plots in 2-DE maps were abundant,dispersed and clear,suggesting that the 2-DE technique developed in this study could absolutely meet the requirement of further proteomics study.We had used 2-DE and matriassisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF/MS) to identify the proteins.From 2-DE data,9 resolved differentially expressed proteins were detected in intracellular proteins.Selected spots were subjected to tryptic digestion followed by identification using MALDI-TOF/MS.We determined 8 peptide mass fingerprinting(PMF) respectively in intracellular proteins by searching in MASCOT database.Eight interrelated proteins expressed differently were identified in intracellular proteins and 4 of them might be involved in PHB's anabolism directly.