Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; metabolism

Objective To evaluate the effect of fast-track TKA upon the recovery time and degree of satisfaction of the patients.Methods 208 patients with primary osteoarthritis between 2013 and March 2015 were randomly divided into 2 groups.In fast-track TKA group was established in peri-operative analgesia,blood management,postoperative anticoagulation,surgical skill and mitigation of perioperative stress.In TKA group,conventional surgical management mode was followed.Identical discharge standards were established for patients in 2 groups to evaluate the recovery time.The patients' degree of satisfaction at postoperative 2 weeks was assessed by numerical rating scale (NRS).The degree of satisfaction about acquisition of preoperative information,degree of satisfaction about postoperative analgesia and postoperative function were statistically compared between 2 groups.Postoperative complication,blood transfusion rate,infectious events within postoperative 3 months,thrombosis-related events,re-operation and death were recorded.Results In fast-track TKA group,the mean discharge time was (2.3±1.2) d,significantly shorter compared with (4.7±5.1) d in TKA group.The mean NRS score in fast-track TKA group was 9.8±0.5 and 9.1±1.4 in TKA group.In fast-track TKA group,the mean scores of the degree of satisfaction about acquisition of preoperative information,degree of satisfaction about postoperative analgesia and postoperative function were 27.7±7.3,9.8± 1.7 and 9.6± 1.3,significantly higher than 15.5±10.1,9.2±2.2 and 8.7±1.8 in the TKA group.In fast-track TKA group,postoperative blood transfusion rate was 3.9％,considerably lower than 11.4％ in TKA group.Conclusion Fast-track TKA can shorten the length of hospital stay,not increase the mortality rate and postoperative complication,accelerate the recovery of daily activity and effectively enhance the degree of satisfaction of the patients.

Objective To investigate the clinical efficacy of PPH and vaginal repair to strengthen recto-cele in the treatment of ODS patients. Methods 87 patients with rectocele were divided into and groups A and B according to the principle of randomization. They were treated by vaginal repair and PPH repair respectively. The two groups were compared in term of curative effect and Longo′s ODS score,operation time,intraoperative blood loss,postoperative pain score,the number of analgesics needed,hospitalization time and hospitalization expenses. Results The heal rates together with marked efficacy rate at months 1 and 3 in group B were significantly higher than those of group A(87.5%,90% vs.77.5%,80%,P<0.05).There were no differences in hospital stay and in-traoperative blood loss.Group B was significantly superior to group A in postoperative pain score,number of analge-sics needed and hospitalization expenses.Conclusion PPH is more complete and more effective in treating consti-pation caused by RC.

Immobilization of enzymes is important and widely applied in biocatalysis. Streptomyces platensis gene gox, encoding an extracellular L-glutamate oxidase (Gox), was fused to cellulose binding domain (CBDcex) from Cellulomonas fimi and the recombinant protein Gox-CBD was expressed in Escherichia coli. The fusion protein (Gox-CBD) was immobilized onto microcrystalline cellulose. The preparation conditions, binding capacity, properties and stability of the immobilized enzyme were studied. Under the condition of 4 ℃, for 1 hour, the fusion protein Gox-CBD was able to bind microcrystalline cellulose at a ratio of 9.0 mg of protein per gram of microcrystalline cellulose. Enzymatic properties of free and immobilized L-glutamic oxidase (Gox-CBD) were compared. The specific activity of the immobilized enzyme decreased, but its thermal stability increased a lot compared with that of the free Gox-CBD. After incubation at 60 ℃ for 30 min, 70% of the total activity remained whereas the free recombinant Gox completely lost its activity. The immobilized protein was tightly bound to microcrystalline cellulose at pH below 10 or more than 5 mmol/L NaCl. The fusion protein of Gox-CBD can be specifically immobilized on the microcrystalline cellulose on a single step. Therefore, our findings can provide a novel strategy for protein purification and enzyme immobilization.