ObjectiveTo study the efficient expression conditions, purification, and partial enzymatic properties of His-tagged recombinant Candida utilis uricase.MethodsThe effects of isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose as inducers which were added in the end logarithmic phase were compared by the method of shake flask culture. The induction culturing time was studied with 50 L fermentor. The protein of interest was purified by Ni-Sepharose and Sephacryl S-200 HR chromatographies, and the optimal pH value, temperature, and thermal stability were also studied.ResultsThe shake flask culturing experiment results showed that IPTG was better than lactose as an inducer. In the fermentor culturing and at the end of the logarithmic growth stage, the enzyme activity of 164 U per gram bacteria and biomass of 23 grams per litter fermented solution were maximal after adding lactose for 7 hours as an inducer, i.e. the enzyme activity could be collected at 3 772 U per liter. The specific activity of purified uricase was 4.5 U/mg and the optimal pH value and temperature were 7.5 and 40℃. Additionally, the enzyme was stable at pH 6.0–10.0 and the thermal stability was below 45℃.ConclusionsIt is better to use lactose as an inducer in the process of culture. The recombinant uricase with His label can be purified by Ni-column affinity chromatography and Sephacryl S-200HR molecular sieve chromatography, and the purification process is easier than ever before. The optimum temperature, pH value, acid-base stability and thermal stability of the purified enzyme have been established to provide some experimental basis for the relationship between structure and function and practical application in the future.
Objective To investigate the value of extravascular lung water index ( EVLWI) and intrathoracic blood volume index ( ITBVI) monitoring in fluid management of severe pneumonia patients with sepsis shock.Methods A prospective controlled study was conducted in106 patients who were diagnosed as severe pneumonia with sepsis shock in intensive care unit fromJanuary 2010 to February 2013. 54 patients who received pulse indicator continuous output ( PiCCO) monitoring were enrolled into the EVLWI + ITBVI group, and EVLWI and ITBVI were used as indicator of fluid management. 52 patients who received central venous pressure ( CVP) as indicator of traditional fluid managementwere enrolled into the control group. The time and the rate to achieve early goal-directed therapy ( EGDT) target were compared between two groups. Acute physiology and chronic health evaluation Ⅱ ( APACHE Ⅱ ) , sepsis related organ failure assessment ( SOFA) , noradrenaline dosage, serumlactic acid, serum creatinine were compared between 1 day and 3 days after treatment. The characteristics of fluid management were recorded and compared within 72 hours. Mechanical ventilation ratio, duration of mechanical ventilation, ICU stay and 28-day mortality were compared between two groups. Results The ratio of achieving EGDT target in 6 hours was significantly higher in the EVLWI + ITBVI group than that in the control group ( 75.9% vs. 55.7% , Plt;0.05) , whereas the time and the ratio to achieve EGDT target in 24 hours were not statistically different. APACHE Ⅱ, SOFA, norepinephrine dosage, serum lactate were significantly decreased 3 days after treatment in the EVLWI + ITBVI group, but did not change significantly in the control group. On3 days after treatment, serumcreatinine was increased in the control group, and did not change significantly in the EVLWI + ITBVI group. The fluid intake and fluid balance volume during 0-6 hours period were significantly higher in the EVLWI + ITBVI group than those in the control group ( Plt;0.05) , but showed no difference ( Pgt;0.05) in other periods. Mechanical ventilation ratio, duration of mechanical ventilation, ICU stay and 28-days mortality were significantly lower in the EVLWI + ITBVI group compared with the control group ( Plt;0.05) . Conclusion Compared with CVP, ITBVI and EVLWI can more accurately assess and guide fluid management in severe pneumonia patients with septic shock with less duration of mechanical ventilation, ICU stay and mortality.