| The cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), one of the representative compatible solutes with the characteristic of zwitterions, accumulates in halophilic and halotolerant bacteria. Previous studies indicated that ectoine functions on proteins, nucleic acids and cells as a stabilizer against some adverse conditions. The produciton of ectoine and its bioprotective application and mechanism were studied in the paper using phytase and lipase as model protein.A moderately halophilic bacterium B2 was isolated from silt in salt lake.'H-NMR analysis was used to identify ectoine in cell extract. Based on the analysis of 16S rDNA, strain was identified as Halomonas (Halomonas sp.). When B2 was cultivated in MG medium with 2 mol/L NaCl, the maximum ectoine concentration was 3.5 g/LThe protective effects of ectoine on thermal stability of phytase (PHYAⅡwas investigated by comparing with other compatible solutes. The concentration, protective mode and effect on pyrohydrolysis of ectoine were studied. The result showed that ectoine had a highest thermoprotection efficiency for PHYAⅡas thermalstability protective additive.According the analyses of thermodynamics and kinetics of heat inactivation of PHYA II, combining with UV and fluorescence spectra, the mechanism of improving enzyme thermal stability by ectoine was speculated. The result indicated that the addition of ectoine improved△U*,△H*,△G* and t1/2, as well as reduced constant k of inactivation rate. The fluorescence and UV spectra showed that ectoine was disadvantaged for stretching of peptide chain after enzyme heat treatment. Therefore, it was suggested that addition of ectoine mainly led to change of hydrogen bond either inside enzyme or between enzyme and water in media, whereas benefit for increasing of the thermal stability of enzymes.Effect of ectoine on Novozym435 and whole cell lipase from Aspergillus oryzae DM-01 were investigated using transesterification for biodiesel and esterification for aethylis oleas as model reactions in organic media. The result indicated lipase would be inhibited by excess metnanol (alcohol) as substrates. Ectoine exhibited significant protection on enzymatic activity in once or reuse. Compared with other compatible solutes, ectoine showed the best protection. However, excess ectoine would result in inhibitting enzymatic activity.Using Fourier transform infrared spectroscopy (FTIR), combined with enzymatic kinetic analysis, the mechanism of improving activities of lipase in methanol (ethanol) and oleic acid by additine of Ectoine was investgated. Original infrared spectra and secondary structure analysis of Novozym435 showed that optimal ectoine could reduce the hydrogen bond association of methanol on enzyme and increase stability of enzyme by increasingβ-sheet structure. In addition, enzyme catalysis kinetics also shows that supplementation with an appropriate amount of ectoine led to a decrease in the affinity of lipase for methanol. The infrared spectrum of A. oryzae DM-01 lipase showed that oleic acid and absolute ethanol had an effect on enzyme conformation, in which oleic acid significantly decreased intramolecular hydrogen bonding to impact on enzyme conformation on greater degree. Addition of ectoine reduced the change trend of secondary structure, which closed to the natural conformation of the enzyme.
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