Heterologous expression and enzymatic properties of the alginate lyase gene vralg1 from Vibrio sp.DS32
Cloning,heterologous expression and enzymatic properties were studied using the alginate lyase gene vralg1 obtained from Vibrio sp.DS32 from the sediment samples of Dongshan Bay,Fujian Province.The alginate lyase gene vralg1 was cloned from DS32 genome and the recombinant expression vector pET-vralg1 was constructed and heterologously expressed in E.coli.The enzymatic properties,substrate specificity and complete degradation products of the recombinase were determined.The optimum temperature of the recombinant enzyme VRALG1 was 35 ℃ and the enzyme activity reached more than 80％in temperatures of 5～50 ℃.The optimum pH was 6.5～7.5 and the enzyme activity was above 90％after incubation at pH 6.0～9.0 for 1 h.The maximum reaction rate of the recombinant enzyme was 5.919 mmol/(L·min),the Michaelis constant was 3.712 mmol/L and the specific en-zyme activity reached 5.874 U/mg under optimal conditions.K+,Cs+,Na+,imidazole and ethanol had little effect on the enzyme activity and the enzyme activity remained above 90％at 5 mmol/L(50 mg/mL)concentration.EDTA had obvious inhibitory effect on the enzyme,and the enzyme was completely inactivated at 1 mmol/L con-centration.VRALG1 had high degradation activity for sodium alginate and PolyG.TLC analysis showed that it was mainly a mixture of monosaccharide,disaccharide and trisaccharide.Combined with substrate specificity analysis,it was speculated that the recombinant enzyme was an endo-type bifunctional alginate lyase with obvious polygulu-ronic acid preference.The alginate lyase gene was successfully cloned and heterologously expressed in E.coli.The obtained recombinant enzyme VRALG1 had excellent sodium alginate degradation activity and obvious polyguluronic acid preference,which could be used to prepare alginate oligosaccharides with low degree of polymerization.
marine biologyalginate lyasealginate oligosaccharidecloning and expressionenzymatic propertypolygururonic acid preference
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维普
