Novel Detection Method for Evaluating the Activity of an Alkaline Serine Protease from Bacillus clausii.

Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the "real" protease activity. Here, a rapid and simple method for determining the "real" activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.

[Posterolateral minimal incision, poking reduction, and simple internal fixation in treatment of collapsed fractures of posterolateral tibial plateau].

Objective: To explore the effectiveness of limited incision, poking reduction, and simple internal fixation in the treatment of collapsed fractures of the posterolateral tibial plateau. Methods: Between October 2010 and January 2016, 16 patients with collapsed fractures of the posterolateral tibial plateau underwent posterolateral incision, poking reduction, and simple internal fixation. There were 10 males and 6 females with the age of 22-63 years (mean, 43.5 years). The injury was caused by falling in 5 cases, traffic accident in 7 cases, and falling from height in 4 cases. All cases had closed fractures. The left knee was involved in 9 cases and the right knee in 7 cases. The injury-to-admission time was 2 hours to 3 days (mean, 10 hours). X-ray films showed that the articular surface collapsing was more than 2 mm. According to Schatzker criteria, 6 cases were rated as type II and 10 cases as type III. Twelve cases had fracture of fibular head. The incision length, operation time, intraoperative blood loss, and incision healing were recorded; fracture healing was observed, and tibial plateau angle and posterior slope angle were measured on X-ray films; loss of articular surface reduction was observed by CT scan; and American Hospital for Special Surgery (HSS) score was used to evaluate the knee joint function. Results: The incision length was 7-10 cm (mean, 8.6 cm); operation time was 35-55 minutes (mean, 46 minutes); intraoperative blood loss was 10-35 mL (mean, 28 mL). Primary healing of incision was obtained. Skin pain occurred in 1 case at 2 months because Kirschner wire retracted. Fifteen cases were followed up 8-21 months (mean, 13.5 months). The fracture healing time was from 3 to 6 months (mean, 4.8 months). There was no significant difference in tibial plateau angle and posterior slope angle between at immediate after operation and at last follow-up ( t=-1.500, P=0.156; t=-1.781, P=0.097). The anatomic reduction rate of articular surface was 93.8% (15/16) at immediate after operation. At last follow-up, the recollapse height of articular surface was 0.1-1.2 mm (mean, 0.36 mm). According to the HSS score system, the results were excellent in 12 cases, good in 2 cases, and fair in 1 case, and the excellent and good rate was 93.3%. Conclusion: The limited incision by posterolateral approach, poking reduction, and simple internal fixation have the advantages of small injury, full exposure, and easy operation in the treatment of simple posterolateral tibial plateau fractures; bone graft support and simple internal fixation can prevent recollapse of the articular surface and achieve satisfactory knee function.