Gene Cloning,expression,characteristics And Application Of Glycerophosphodiesterase From Bacillus Altitudinis W3

Glycerophosphodiesterases（GDPDs）（EC 3.1.4.46）,which are widely expressed in prokaryotic and eukaryotic organisms.It can decompose glycerol phosphodiester into glycerol-3-phosphate（G3P）and corresponding alcohols,and plays an important role in the metabolism and synthesis of phospholipids in organisms.In this paper,a GDPD gene（bagdpd）was screened from B.altitudinis W3,which was successfully cloned and expressed in E.coli BL21（DE3）.The fermentation conditions were optimized,and the enzymatic properties of the recombinant enzyme and its application in DPHP degradation were studied.The main research results were showed as follows:（1）A GDPD gene（bagdpd）was screened from the whole genome of B.altitudinis W3.The enzyme was determined to be a member of the GDPD superfamily through bioinformatics analysis.The gene is 738 bp in length and encoded a total of 245 amino acids,with a theoretical molecular weight of 28.0 k Da.Ba GDPD was closely related to GDPD from Bacillus safensis（Gen Bank No.ayj89117.1）.It had a highly conserved sequence motif（HR（X）n EN（X）n EXD（X）n HD）containing active site residues,belonging to the phosphatidylinositol phosphodiesterase superfamily.（2）The recombinant strain was constructed,and the fermentation conditions at the shake flask level of the recombinant bacteria were optimized by the single factor method.The results showed that the optimal fermentation medium was beef extract 7.5 g·L^-1,yeast powder 7.5 g·L^-1,glucose 10 g·L^-1,glycine 0.5%（w/v）.The optimal induction conditions were IPTG concentration 0.4 mmol·L^-1,induction at 15℃for 24 h.After fermentation under the optimal conditions,the enzyme activity reached 34.88 U·m L^-1,and the optimized fermentation enzyme activity was 2.53 times that of the original fermentation enzyme activity.（3）The optimum temperature of rBa GDPD was 55°C and rBa GDPD maintained high activity in the range of 40-60°C.When the temperature was lower than 25℃or higher than75℃,the enzyme activity was very low.The optimum p H was 8.5,and the enzyme maintained high activity in the p H range of 7.0-11.0（its residual activity was more than 80%）.The rBa GDPD was a metal ion dependent enzyme,and could not show catalytic activity for Bp Npp without adding any metal ions or adding EDTA.Ni²⁺had the most obvious activation effect on rBa GDPD,and had the best activation effect on rBa GDPD when the concentration was 50mmol·L^-1.The kinetic parameters of rBa GDPD showed that the K_m value was 3.56±0.20mmol·L^-1,and the k_cat/K_m value was 0.40 L·mmol^-1·s^-1.（4）The degradation effect of rBa GDPD on diphenyl phosphate（DPHP）was studied.The reaction was terminated at 2,4,6,8,10 and 12 h,and the degradation rate was determined by HPLC.After 12 h treatment,the degradation ratio of DPHP reached 71.25%,and the degradation product was identified as monophenyl phosphate（PHP）by LC-MS.