Study On Prokaryotic Expression And Properties Of Thermostable Galactosidase From Bacillus Coagulans

Galactosidase can hydrolyze lactose in dairy and produce low lactose dairy to solve the problem of "lactose intoierance".While normal temperature galactosidase have so many defects in practical production such as low sterilization temperature,low activity,high costs,et al.This paper focused on thermostable galactosidase,which was cloned and expressed from Bacillus coagulans sp.T242 in E.coli BL21/pET32-gal T242.Fermentation conditions and inducement conditions of recombinant E.coli,isolation,purification and characterization of thermostable galactosidase were analysed.It is to establish the foundation for the industrial applications of thermostable galactosidase.In this paper,DNAStar,BLAST and SWISS-MODEL were used to analyse the gene sequence and space structure of thermostable galactosidase.The gene sequende of it from Bacillus coagulans sp.T242,which was filtered in our laboratory,was found having an identity of 100%with a part of genomic DNA of Bacillus coagulans 36D1 in GenBank data base.It was presumed that this galactosidase gene encode 665 amino acids,with protein molecule 76.09kDa.And there were two glucosylation sites:NKSW-181,NHTD-623.Fermentation and induction conditions were optimized of recombinant E.coli.Optimum conditions were:4h age of seed,1.5%inoculation amount,30%liquid medium volume,160 r/min shaking revolution,adding IPTG after culturing for 5h with a final concentration of 0.6mmol/L,induced and cultured for 5h at 37?.In this condition,the activity of thermostable galactase was 5.242U/mL per OD600.DEAE-52 and Sephadex G-75 were used to separate the thermostable lactse from Bacillus coagulans,while Ni-NTA was used to separate the recombinant enzyme.The purification efficiency was 75%.The activity of recombinant thermostable lactase was 4.3 folds of original enzyme.Purification results were analysised by SDS polyacrylamide gel electrophoresis,and its molecular mass was estimated to be about 55.0kDa.Study on enzyme properties of the purified recombinant thermostable galactosidase which took ONPG as substrate were as follows:the optimal temperature of the lactase was 50?,low thermostability higher than 55 ?;the optimum pH of the lactase was 6.8,and it was stable at neutral or meta-acid;when the metal ions concentration was lmmol/L in enzyme reation,Mg2+?Ca2+ had activation on the lactase.Especially Ca2+ had a significant activation,the enzyme activity can be increased to 150%of the blank control.Cu2+?Fe2+ had inhibition on enzyme activity,but not obviously.For ONPG the Km was 2.21mmol/L and Vmax was 0.87mmol/L·min in condition of pH 6.8,50?.