As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^-1 and 898.03 s^-1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.
Laccase/chemistry* ; Directed Molecular Evolution ; Enzyme Stability ; Bacillus pumilus/genetics* ; Mutation ; Biocatalysis ; Catalysis

Objective: To investigate the clinical pathological features of Barrett′s esophagus in China, and to study the relationship between the number of goblet cells and the severity of Barrett′s esophageal dysplasia. Methods: From January 2008 to October 2018, in the Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, the clinical and pathological data of 453 patients who underwent gastroscopy and pathologically diagnosed with Barrett′s esophagus were retrospectively analyzed. The clinical pathological features were compared between patients with goblet cells and patients without goblet cells. Periodic acid Schiff reaction (PAS) staining was performed on pathological slides of Barrett′s esophagus with goblet cells, and the relationship between the number of goblet cells, the number of positive crypts of goblet cells and the severity of Barrett′s esophageal dysplasia was analyzed. T test and chi-square test were performed for statistical analysis. Results: Among 453 patients with Barrett′s esophagus, 251 (55.4%) were males and 202 (44.6%) were females. There were 218 Barrett′s esophagus with goblet cells, including 128 males (58.7%) and 90 females (41.3%). The average onset age was (60.6±11.9) years old, and the peak onset age was between 60 and 69 years old. The appearance under endoscopy mainly was circumferential type (58.2%, 127/218). There were 235 Barrett′s esophagus without goblet cells, 123 males (52.3%) and 112 females (47.7%). The average onset age was (56.1±14.4) years old, and the peak onset age was between 50 and 59 years old. The appearance under endoscopy was mainly circumferential type (40.0%, 94/235). The incidence of dysplasia in Barrett′s esophagus with goblet cells was higher than that without goblet cells (75.7%, 165/218 vs. 37.0%, 87/235), and the difference was statistically significant (χ²=68.501, P<0.01). PAS staining showed that goblet cells were stained purplish red. The number of goblet cells, total number of crypts, the number of positive crypts of goblet cells and the proportion of positive crypts of goblet cells of Barrett′s esophagus with mild dysplasia were all significantly higher than those of Barrett′s esophagus with moderate dysplasia (95.50±40.56 vs. 40.00±13.34, 21.00±8.31 vs. 11.83±2.92, 16.50±6.17 vs. 7.50±2.47 and 0.79±0.42 vs. 0.63±0.12, respectively), and the differences were statistically significant(t=-4.503, -3.605, -4.690 and -4.340, all P<0.01). Conclusion Barrett′s esophageal dysplasia may be related with appearance of goblet cells, and the decrease or disappearance of goblet cells may indicate the progression of Barrett′s esophagus.

Objective To re-evaluate the potential risk of small gastric stromal tumor ( diameter less than 2 cm), and to assess the safety and efficacy of endoscopic resection and further treatment strategy for small gastric stromal tumor. Methods Data of 584 patients undergoing endoscopic submucosal dissection ( ESD) and diagnosed as gastric submucosal tumor ( SMT) in Tianjin Medical University General Hospital from September 2008 to December 2015 were retrospectively analyzed. The clinical and pathological features and potential risk of small gastric stromal tumor were analyzed. The therapeutic outcomes, complications and follow-up results of ESD were summarized, and the follow-up results of patients undergoing ESD were compared with 45 patients who were suspected as small gastric stromal tumor and followed-up regularly by endoscopic ultrasonography ( EUS) in the same period. Results Among 584 cases of gastric SMT, stromal tumor ( 239 cases, 40. 9％) was the most common type, of which small gastric stromal tumor was found in 203 cases (84. 9％, 203/239). The mitotic index of all cases was no more than 5/50 high power field. However, adverse factors under EUS, mainly including strong echo and heterogeneity, were reported in 94 (46. 3％) out of 203 cases and were confirmed to be related to tumor size (P=0. 000). ESD was successfully performed in 203 patients with small gastric stromal tumor, and no serious complication or perioperative death occurred. Symptoms of 81. 4％ ( 144/177 ) patients were improved after ESD, and no stromal tumor recurrence or metastasis was observed during a follow-up of 12-84 months. Of the 45 patients followed-up regularly by EUS, 38 (84. 4％) patients had gastrointestinal symptoms and 84. 2％ (32/38) were not relieved during follow-up, and 12 (26. 7％) had a heavier psychological burden, seriously affecting the quality of life. Conclusion The incidence of adverse factors under EUS is high in patients with small gastric stromal tumor, and increased with tumor size. ESD is safe and effective for small gastric stromal tumor, which contributes to the diagnosis, further improves therapeutic effects and reduces psychological pressure.

To investigate the safety and clinical efficacy of endoscopic submucosal dissection (ESD) in the treatment of gastrointestinal neuroendocrine neoplasms (GI-NENs). Methods: The clinical and pathological features of 39 patients with GI-NENs who were treated with ESD, the efficacy of ESD treatment, complications, and follow-up results were analyzed retrospectively. Results: The 39 cases of GI-NENs were all neuroendocrine tumors (NETs). According to histopathology and proliferative activity, there were 37 cases of G1, two cases of G2, and no cases of G3, including 23 cases of cancer in the rectum, 12 in the stomach, 4 in the duodenum. Thirty-five lesions invaded the mucosa or submucosa, four invaded the muscularis propria, one perforated in the ESD, and two were associated with bleeding. After 6 to 90 months of follow-up, new lesions were found in 1 patient with g-NETs in different parts of the stomach at 5 and 34 months after operation, followed by ESD treatment and further follow-up. No lymph node and distant metastases were found, and the survival rate of the patients was 100%. Conclusions: For GI-NENs without lymph node and distant metastases, the lesion is con-fined to the submucosa, and a diameter≤1.0 cm is an absolute indication of ESD. For rectal neuroendocrine neoplasms that have non-invasive muscularis propria with diameters between 1 and 1.5 cm, or for patients with Type 1 gastric neuroendocrine tumors (T1-GNETs) that are predicted to be T2, ESD treatment should be prioritized to preserve gastrointestinal volume and function.