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Phosphatidylserine synthase belong to the phosphatidyl diesters synthesis enzymes, with the obvious features of this family, i.e. H (x) K (x) 4D contains two spaced appears conserved sequence. The phospholipid phase catalytic substrate material and the aqueous phase nucleophilic donor transesterification reaction similar to the ester condensation is of synthetic phosphatidylserine rare phospholipids good tools enzyme. With a high degree of specificity of the enzyme conversion and modified on a wide range of existing sources of crude phospholipids, can be prepared rare phospholipids, and the field of health care products in the pharmaceutical and food with great value. Bacillus subtilis was transformed by PCR technique to the size of a DNA fragment of 1350bp encoding phosphatidylserine synthase (ExPASy P23830) was amplified from Escherichia coli K12, and insert it into the Bacillus subtilis-inducible expression vector pBES recombinant plasmid pBES-pss after induction the DB104o of sucrose, the phosphatidylserine synthase guide to the extracellular secretion expression in Bacillus subtilis DB104 (pBES-pss), SDS-PAGE analysis revealed that the molecular weight of the target protein of approximately 53KDa, enzyme-linked colorimetric detection of enzyme activity was 1.50U / mL. Use of 250 mL flask fermentation engineering bacteria carrying phosphatidylserine synthase gene recombinant plasmid were induced to study the pH of the medium, the liquid volume, speed, and during the induction of cell concentration, concentration of inducing agent, the induction time and The temperature on the phosphatidylserine synthase activity. Determining the engineering bacteria optimized fermentation conditions were: a medium pH of 7.5, a liquid volume 40 mL, the rotational speed of 200 r / min, during the induction of cell concentration of OD600 of ≈ 0.4, the inducing agent sucrose final concentration of 60 mmol / L, the induction time of 27 h, the induction temperature of 37 ° C. Optimum fermentation conditions, the activity reached 2.56 U / mL, is before optimization 1.72 times. pBES-pss fermented crude enzyme solution by ammonium sulfate precipitation, concentrated by the hollow fiber membrane desalination, SP-Sepharose HP ion exchange chromatography and Sephadex G-75 gel chromatography, basic obtain electrophoretically pure recombinant phosphatidyl serine synthesis enzyme, the specific activity of up to 13.62U/mg, is 39.59 times before purification. Enzymatic Properties: enzyme catalyses the of lecithin hydrolysis reaction of the optimum pH was 8.0, the optimum temperature of 35 ° C. Stability studies show that: the enzyme in the pH 6.5 to 9.5 range and is stable under the temperature below 45 ° C. Various surface-active agents and metal ions affect the results show that the hydrolysis activity of the enzyme: SDS, Tween 20, Tween80, the enzyme inhibition, Triton X-100 enhanced the enzyme; Mg2, Zn2, K of the enzyme inhibition, Ca2, Mn2 and EDTA enhanced role of the enzyme.
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