Breeding Of Twin Enzyme-producing Pseudomonas Putida And Enzymatic Process For D-pHPG

D-hydroxyphenylglycine (D-pHPG) is the most important intermediate used for the synthesis of Semisynthetic antibiotic including amoxicillin and Cefadroxil. The two main synthesis methods for D-pHPG are chemical processes and enzymatic processes, among which the process using one strain with twin enzyme is the best for the environmental and economic concerns. Pseudomonas putida studied in this paper can produce both D-Hydantoinase (DHase) and D-Carbamoylase (DCase) in its growing period. Twin enzyme (DHase and DCase) can catalyse DL-p-hydroxyphenylhydantoin (DL-pHPH) to D-pHPG directly.The main contents of this paper were:to identify the strain that can produce twin enzyme and produce the intermediate N^carbamoyl-D-p-hydroxyphenylglycine (NC-D-pHPG); to mutate and screen the strain with ultraviolet radiation(UV) and diethyl sulfate(DES); to mutate and screen the strain with N+ion implantation; to optimize the culture condition of mutated strain; to isolate and purify twin enzyme and immobilize DHase; to produce D-pHPG with immobilized DHase and purified DCase.With the identification of the strain spMF0507, it was determined that its logarithm growing period was about 20-50hours; the best growing temperature is 28-32℃, the best enzyme producing temperature is 28-30℃; The best growing pH was 6.5-7.5. The best enzyme producing pH was 7.0-7.5. DHase and DCase showed different activity under different temperature and pH. DHase was more stable than DCase. DCase will be inactivated by being heated at 55-60℃for about 30 min. DL-pHPH can translate to NC-pHPG by reacting with the strain of inactivated DCase under following conditions:DL-pHPH concentration 6%, temperature 33℃and pH9.0-9.5. The crystallized rate of NC-D-pHPG was 97.9% and the purity of NC-D-pHPG was 98.5% by HPLC.A high twin enzyme producing, strain spUD0612 with genetic stability was obtained from Pseudomonas putida spMF0507 by mutagenesis with UV 30s and diethyl sulfate for 20min,followed by resistance selection with substrate analogue 20μg/mL 5-FU. After the screening of fermentation with a shake flask, its activity of DHase and DCase reached 1.784 U/mL and 0.865 U/mL, respectively. Compared with starting strain, the enzyme activity enhanced 87.39% and 58.42%, respectively. A high twin enzyme producing strain spUDN0706 with genetic stability was obtained from Pseudomonas putida spUD0612 by mutagenesis with N+ ion implantation (30KeV,8×1015ions/cm2,10-3Pa,10mA,8s)followed by resistance selection with substrate analogue 25μg/mL 5-FU. After the screening of fermentation using a shake flask, its. activity of DHase and DCase reached 2.482 U/mL and 1.253 U/mL, respectively.Compared with spUD0612, the enzyme activity enhanced 46.69% and 53.93%, respectively.The culture medium and the fermentation conditions were optimized based on the former culture medium and formal fermetation conditions. The results of the experiments showed the best fermentation conditions were as following:inoculum amount 8-10%, loading amount 100mL,culture temperature 30℃, initial pH7.5.And the best composition were as following:corn steep liquor 2.0%,glucose 2.5%, revulsent 3.5%,NaCl 0.3%,MgSO40.05%,CoCl20.01%,(NH4)2SO40.1%. The spUDN0706 grew well on the optimized medium and fermentation condition.Its activity of DHase and DCase reached-2.591U/mL and 1.622U/mL, respectively. Compared with the formal medium and fermentation condition, the enzyme activity enhanced 14.04% and 28.63%, respectively. By validating on 5L auto-fermentor, its activity of DHase and DCase reached 3.078U/mL and 1.953U/mL, respectively. Compared with using shake flask, the enzyme activity enhanced 18.80% and 20.41%, respectively.Collecting the strain of spUDN0706 fermentation broth and breaking up the strain making use of homogenizer, the best pressure was 70MPa and the best breaking up time was 15min. Purifying twin enzyme with 20%(NH4)2SO4 and 34%(NH4)2SO4 can isolate and purify both DHase and DCase. After flocculation, purification and ultrafiltration, the total purified yield and purification folds of DHase were 69.2% and 6.4, respectively; and the total purified yield and purification folds of DCase were 76.4% and 5.45, respectively. The effects of some parameters on DHase immobilization were investigated, and the optimized conditions were obtained as follows:carrier TJS input 133U/lg, immobilization time 15h, temperature 28℃, pH7.5 and the activity of immobilized enzyme was 48.7U/g, the immobilization yield was 34.8% under the optimized conditions.By checking the activity of immobilized DHase and purified Dcase, it was determined that the best pH and temperature for immobilized DHase were 9.5 and 60℃respectively; and the best pH and temperature for DCase were 7.5 and 40℃, respectively. Immobilized DHase was more stable than purified DCase against temperature and pH. Ions including Mn2+, Ca+, Mg2+, Ba2+could activate both immobilized DHase and purified DCase.Co2+activated immobilized DHase but inhibited purified Dcase. Fe2+, Zn2+, and especially Hg2+inhibited both immobilized DHase and purified DCase. The half life of immobilized DHase was 210 days and its apparent Km was 20.14mmol/L; the half life of purified DCase was 6 days and its apparent Km was 8.40mmol/L. Immobilized DHase and purified DCase can catalyse the conversion of DL-pHPH to D-pHPG in one step under following conditions: substrate conc.3%,temperature 38℃,immobilized DHase input 5kU/L,purified DCase input 3kU/L. Immobilized DHase could be used over 256 cycles and conversion rate and yield were all over 97% averagely. After purification by ultrafiltration and concentration, the content of the translated D-pHPG was 99.2% and its polarity was-159.8°, achieving a satisfactory reliability.