Purification And Biochemical Characterization Of An Esterase BSE-1 From Marine Bacillus SP.

Marine Bacillus sp. esterase BSE-1 was secreted by marine Bacillus sp.. The dissertation focused on the screening and identification of the strains, separation and purification of BSE-1, and its properties, catalytic kinetic, thermal inactivation dynamics etc. The research will make a basis to its further structure-functional study, its industrialization and application.Based on the fundamental principle and speciality of the microbial esterase, proper substrates (Tributyrin) and indicators (Rhodamine B) were used to screen the esterase-producing bacteria in the preliminary and secondary screening. A precise and rapid screening method was developed. The strain of EB-1, which was isolated from related marine soil samples, showed the highest esterase-producing ability (22.8 U/ mL). The strain EB-1 was identified as Bacillus subtilis var. niger based on the analysis of the morphological, culture, physiological biochemical characteristics and the sequence of 16S rDNA. BSE-1 was induced from intracellular enzyme to extracellular enzyme by adding 0.5% olive oil and 0.5% glycerol in the culture medium.SDS-PAGE homogeneity BSE-1 was purified from the fermentation liquid by high speed freezing centrifugation (10000r/min, 30min, 4℃), ethanol-ammonium sulfate aqueous two-phase precipitation and Q-Sepharose HR anion exchange chromatography. SDS-PAGE showed its molecular weight was 30 kDa. In further studies, with PNPP as substrate,the results showed that the optimum pH of this esterase was 10.0 and its optimum reaction temperature was 60℃. BSE-1 was stable when the temperature was below 60℃and pH among 7~11. This character gave BSE-1 good property to apply under higher temperature. BSE-1 also showed high activity at rather higher alkaline pH (higher than 10). BSE-1 was compatible with some acid radical ions and showed good resistance to general organic solvent. The effects of metal ions on BSE-1 showed that Li+,Mg2+,Ca2+ could increase its activity. On the other hand, Ba2+,Fe3+,Ag+,Sr2+ inhibited the activity of BSE-1. The inhibition of EDTA on BSE-1 was remarkable, which suggested there were metal ions in the active centre of BSE-1.The catalytic kinetics and thermal inactivation kinetics of a novel alkaline esterase BSE-1 were studied with the PNPP as substrate. The behavior of the BSE-1 on PNPP hydrolization followed Michaelis- Menten kinetics,with Km= 8.15 mmol·L-1 and Vmax=0.97 mmol·mg-1·min-1. The effect of Ca2+ on the enzyme was found to be of mixed activation type. The inhibition of Ba2+ on the enzyme appeared to be partial noncompetitive type. In addition,thermal deactivation kinetics of BSE-1 was analyzed by continuous model under the temperature of 70℃. Suitable mechanism formula was proposed to describe the inactivation process. And the constants of thermal inactivation rate were k1=1.41 and k2=0.28.