Biotechnological Production Of 3-Hydroxypropionaldehyde By Microbial Glycerol Dehydratase

3-hydroxypropionaldehyde（3-HPA） is an effective antimicrobial agent and a precursor of a series of chemicals such as acrolein and 1,3-propanediol,which made it an important platfrom compound in chemical industry.Biotechnological production of 3-HPA has several advantages over the chemical process such as excellent specificity,mild conditions and eco-friendly.A colorimetric screening method was established based on the detection of 3-HPA by its reaction with 2,4-dinitrophenylhydrazine （DNPH）.Six microorganisms were isolated through the method.Based on morphology,physiological tests and 16S rRNA sequence analysis,one of these strains numbered ZJB-09105 was identified as Lactobacillus.reuteri.The fermentation medium and conditions of L.reuteri ZJB-09105 were optimized by single factors and response surface methodology（RSM）. The optimized conditions for cell growth and formation of glycerol dehydratase were as follows:initial pH value,6.2;37℃;inoculum volumn,4%（v/v）.The optimized medium composition was as follows（g/l）: peptone 10,beef extract 10,yeast extract 14.46,lactose 14,sodium acetate 5,Tween-80 1,ammonium citrate 4.59,K₂HPO₄ 2,MgSO₄·7H₂O 0.2, MnSO₄·2H₂O 0.15.Under these conditions,a specific glycerol dehydratase activity of 1.191 U/mg_DCW was achieved after cultivation for 17 h,which was 95%higher than that obtained under intitial conditions.Influences of reaction conditions on glycerol dehydratase activity were also investigated.The results indicated that the glycerol dehydratase exhibited maximal activity in phosphate buffer（pH 5.91） at 35℃,and the glycerol dehydratase remained high activity under partial acid conditions. Under the optimal conversion conditions,the accumulation of 3-HPA reached 4.2 g/l after 2 h.Finally,to improve the reuse of biocatalyst,alginate was employed to immobilize L.reuteri ZJB-09105.Reaction conditions of immobilization cell-mediated conversion were evaluated.The results showed that optimal working pH of the glycerol dehydratase ranged from 4.6 to 5.0,and temperature ranged from 35 to 37℃.The half-life(t_1/2) of glycerol dehydratase in immobilized cells(t_1/2 27.2 min) and free cells(t_1/2 20.2 min) were determined,which suggested that the thermostability of the glycerol dehydratase of L.reuteri ZJB-09105 was low.Under the optimized conversion conditions,the accumulation of 3-HPA was limited after 3.5 h. After eight runs,the immobilized cells just retained half of its original activity.