To improve bond selectivity of recombinant β-glucuronidase in Escherichia coli (PGUS-E), based on the PGUS-E structure guidance, three key points R329, T369 and N467 were identified to be responsible for the bond selectivity of PGUS-E, and further saturation mutagenesis was conducted. Two positive mutants R329K and T369V were obtained by a combined selection technique of thin-layer chromatography and high performance liquid chromatography. Compared to PGUS-E, the bond selectivity of mutants R329K and T369V increased by 26.9% and 34.3%, respectively; whereas the biochemical properties such as pH and temperature profile were unchanged. Nevertheless, the activity was decreased compared to PGUS-E. These results further confirmed that sites R329 and T369 played important roles for the bond selectivity and activity. In summary, this study significantly increased the bond selectivity of PGUS-E by structure guided saturation mutagenesis, providing experimental support for elucidating the relationship between the structure and function of PGUS-E.
Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Escherichia coli ; metabolism ; Glucuronidase ; chemistry ; Industrial Microbiology ; Mutagenesis ; Recombinant Proteins ; chemistry ; Temperature

Objective: To investigate the variants of middle colic artery (MCA) and ileocolic vein (ICV) and their influence on the deci-sion regarding approach of laparoscopic right hemicolectomy. Methods: We analyzed the diagnosis and treatment of one right colon cancer patient with variant MCA and ICV who was admitted to the Tianjin Medical University Cancer Hospital in March 2018. The pa-tient underwent laparoscopic right hemicolectomy via a limited medial approach after a multidisciplinary treatment (MDT) discussion. Following were the observation indicators: 1) surgical and postoperative recovery situations; 2) postoperative pathological examina-tion; and 3) follow-up situation. Results: 1) Surgical and postoperative recovery situations: the patient successfully underwent laparo-scopic right hemicolectomy via a limited medial approach. No intraoperative or postoperative complications occurred. Duration of postoperative hospital stay was 11 days. 2) Postoperative pathological examination: the number of dissected lymph nodes was 39. Postoperative pathological tumor stage was pT3N0. Postoperative pathological tumor type was moderately differentiated adenocarci-noma. 3) Follow-up situation: the patient was followed-up for 10 months with disease-free survival. Conclusions: Individual and stan-dard surgery will be the best choice for treating colon cancer patients. MDT can facilitate clinical decision-making and benefit patients.