Identification Of Compatible Solutes And Study On Lipopolytic Of Chromohalobacter Sp. From Moderately Halophilic Bacteria

With the development of production technology,enzymes play an increasingly important role in industrial production.It is inevitable that there will be salt environment in the industry.In order to make the enzyme still play a role in the salt environment,it is particularly important to look for the enzyme with salt-tolerance.Halophilic bacteria have the potential to produce enzyme with salt-tolerance.In this study,halophilic bacteria S2 was isolated from the brine of ancient salt well of Zigong,China.The results showed that S2 could produce esterase and lipase.The strain S2 was identified as Chromohalobacter canadensis strain by 16S rRNA.The salt adaptation mechanism of moderately halophilic is to resist the salt environment by the compatibility osmosis mechanism.Compatible solutes are small molecular organic compounds that are compatible with the cellular system without affecting the function of other macromolecules.Compatible solutes are polar in the range of physiological pH,free of charge and soluble.High performance liquid chromatography?HPLC?and mass spectrometry?MS?were used to determine the salt tolerance mechanism of ectoine as the compatible solute.According to the known sequence of Chromohalobacter esterase gene in GenBank,EST gene was successfully amplified from halophilic strain S2.GenBank number was MF769788.The nuclcic acid sequence of EST was 646 bp,encoding 213 amino acid residues.The acid sequence analysis showed that EST was a new family of esterase.EST was inserted into the expression vector pET28a and transformed into E.coli BL21?DE3?.Soluble protein were induced by IPTG with final concentration of 250?mol/L at 16?,150 r/min.The recombinant protein was affinity purified by nickel ion chelating resin,and the purified esterase was named EstS2.The enzymatic properties of EstS2 were analyzed.The results showed that the optimum temperature and pH of EstS2 were 35?and 8.5,respectively,and the enzyme activity could be maintained at a high level in the range of30?⁵0?.When NaCl concentration was 0.25 mol/L,the enzyme activity remained63.1%.With the increase of NaCl concentration,the enzyme activity gradually decreased to zero.In addition,EstS2 has good resistance to most metal ions and chemical reagents.The S2 fermentation broth was purified to obtain a LipS2 with a molecular weight of58 kDa.LipS2 has good hydrolytic activity for triglycerides with carbon chain length greater than 12 and some vegetable oils composed of polyunsaturated fatty acids.LipS2showed high activity in range of 2.5 mol/L to 3.5 mol/L,but no activity without salt.The highest enzyme activity was achieved at 55 C,8.5 pH and 3 mol/L NaCl concentration.Notably,the thermostability and pH stability of LipS2,varying with salt concentration,reached optimum in the presence of 3.0 mol/L NaCl.LipS2 was stimulated by Mg²⁺and Ca²⁺,inhibited by Zn²⁺?Cu²⁺?Mn²⁺?Fe²⁺and Hg²⁺.Moreover,LipS2 displayed significant tolerance to organic solvents including methanol,ethanol,ethyl acetate and acetone,especially,LipS2 activity was enhanced markedly by the hexane and benzene.Non-ionic surfactants increased LipS2 activity,while ionic surfactants decreased activity.