Thermophilic Xanthan Degradation Bacteria Mutation Breeding And Enzyme Production Optimization Research

Xanthan gum is a kind of water-soluble polysaccharide. Due to it good water solubility, unique rheological property and stability of acid and alkali is as an important work in oil drilling fluid thickener. However, it good stability and viscosity brought difficult for processing of crude oil, it is the key process of oil exploration to effective degradation of viscisity. High temperature field generally break with chemical method, this method not only low efficiency but also can bring the serious destruction to environment and geology. In recent years, Bio-enzymatic degradation of xanthan gum is a safe, effective and pollution-free method, it draws widespread attention. This experiment aimed to improve the degrade ability of thermophillic xanthan degradation bacteria HT-1 by DES-UV compound mutagenesis, then excellent mutant strains degradation enzyme production optimization. For oil industry provide applicable engineering bacteria that can degrade the xanthan gum, reduce the viscosity of crude oil, improve the efficiency of mining,mutate xanthan degradation bacterium HT-1 by DES, find it best mutagenesis time is 30 min, screening two genetic and excellent mutant strains, named DE-2 and DE-14, after two days of fermentation cultivation, their viscosity reduction rate respectively were 36.3% and 35.3%,compare with the same batch of experimental strains of HT-1 the viscosity reduction rate increased by 15.0% and 14.3%. Then, mutate this two excellent strains, find their best mutagenesis time is 240s, after screening get the best mutant strains DE-2.3, it viscosity reduction was 44.1%. its improved 22.6% than HT-1.Induced strain DE-2.3 as the research object, optimized the enzyme production medium and fermentation condition through the single factor experiment and response surface experiment. The optimal fermentation medium is (1L):3g Xanthan,3g(NH4)2PO4,0.25g CaCl2,0.25g K2HPO4,0.4g NaCl,0.125g MgSO4,0.35g KNO3; The optimal condition is:temperature 50℃, the speed of 120r/min, pH7.5. After the optimization of culture medium and fermentation condition on DE-2.3 cultivation, shake flask 20h, the strains producing enzyme activity up to 483.7U/LUnder culture conditions optimized for DE-2.3 carried out progressively expanding culture. Respectively in 250mL、500mL and 1L flask expanding culture, the production trends and activity of xanthan degradation enzyme remained the same. Tendency of xanthan degradation enzyme activity held unanimously when expanding culture in 5L fermente, but enzyme activity decreased about 24.3%.