Study On The Bioconversion Of D-p-Hydroxyphenylglycine In Aqueous Two-phase System

D-p-hydroxyphenylglycine（D-p-HPG） is one kind of important intermediate for the preparation of semisynthetic antibiotics, such as semisynthetic amoxicillin, ceforperazone and cefoprozil. In aqueous systems, the solubility of D, L-p-hydroxyphenylhydantoin（DL-p-HPH） is very low. Thus, bioconversion amount is low in unit time and the method of biotransformation using cells was inhibited in large-scale industry. In China, using chemistry syhthesis method to production of D-p-HPG is leading. Also, it is very serious of the pollution of environment. Thus, it is important to select a system to improve the substrate solubility.In this paper, using Pseudomonas putida which can produce D-hydantoinase （D-Hase） was chosen. By experiment, the fermentation condition of fungus were optimized. The partition coefficient of the cells, DL-p-HPH and bioconversion products were studied and using the whole cells to bioconversion were also optimized in aqueous two-phase system （ATPS）. Compared to aqueous system and ATPS, the stability and the reaction ratio of D-Hase were also studied.Based on single factor experiments and orthogonal experimental design, the optimal compositions of fermentation medium were as follows: Glycerol 25 g/L, beef extract 15 g/L, NaH₂PO₄ 10 g/L, KH₂PO₄ 7.5 g/L, MgSO₄ 0.25 g/L, Cocl₂ 0.05 g/L, DL-HPH 1.0 g/L. The optimal culture conditions were as follows: the initial pH of medium 7.0, culture temperature 28℃, inoculation quantity 10%, culture medium cubage 50ml medium in 250 ml shaken flask and rotation rate 220r/min, culture time 24h. The optimal D-Hase activity was 0.7 U/ml (OD₆₀₀=7.0).The study on partition coefficient in PEG/mineral ATPS, the date show that the partition coefficient of the cells including D-Hase was more than 100 and it is propitious to the need of in technics. The partition coefficient of DL-p-HPH owned a large changeable scale in PEG/Na₂SO₄ ATPS. The partition coefficient of CpHPG and D-p-HPG were accessible to 1 in PEG/ Na₂SO₄ ATPS. In optimal 15% （w/v）PEG1000/15% （w/v）Na₂SO₄ ATPS, The partition coefficient of DL-p-HPH, CpHPG and D-p-HPG were 28.6, 1.45 and 1.25, respectively.When resting cells of 0.02g DCW/ml were shaken （220rpm,28℃）in 15% （w/v）PEG1000/15% （w/v）Na₂SO₄ ATPS, 50mM DL-p-HPH as substrate at pH 9.9 and reaction time 24 h, the conversion yield of DL-p-HPH achieved to 61%. Compared to the aqueous system, the solubility improved 10 folds and the converSion amount of CpHPG was 6 folds.The reaction ratio of enzyme in aqueous system is quicker than that in ATPS. After ten hours’ enzymatic reaction, the activity of enzyme disappeared in aqueous system. While, in PEG/Na₂SO₄ ATPS, the activity of enzyme can prolong and still can be bioconversion after twenty hours’ enzymatic reaction.