Enhancing Enzyme Activity and Thermostability of Bacillus amyloliquefaciens Chitosanase BaCsn46A Through Saturation Mutagenesis at Ser196.

Chitosanase plays an important role in chitooligosaccharides (COS) production. We found that the chitosanase (BaCsn46A) of Bacillus amyloliquefacien was a good candidate for chitosan hydrolysis of COS. In order to further improve the enzyme properties of BaCsn46A, the S196 located near the active center was found to be a critical site impacts on enzyme properties by sequence alignment analysis. Herein, saturation mutation was carried out to study role of 196 site on BaCsn46A catalytic function. Compared with WT, the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. In addition, we found that the enzyme activity of S196P was 2.41% of that of WT, indicating that the type of amino acid in 196 site could significant affect the catalytic activity and thermostability of BaCsn46A. After molecular docking analysis we found that the increase in hydrogen bonds and decrease in unfavorable bonds interacting with the substrate were the main reason for the change of enzyme properties which is valuable for future studies on Bacillus species chitosanase.

High preoperative serum ferritin predicted poor prognosis in non-metastatic colorectal cancer.

OBJECTIVES: To validate the prognostic significance of preoperative serum iron metabolism parameters in non-metastatic colorectal cancer patients treated with curative resection.   Methods: We conducted a prospective cohort study in the Department of Surgical Oncology, WuXi 4th People's Hospital, WuxiChina, between  March 2010 and September 2013.  The relationships of serum iron metabolism parameters with other variables were examined. The prognostic significance was evaluated using the Kaplan Meier curve and Cox proportional hazards regression model.  Results: Five hundred and fourteen patients were eligible for analysis. The levels of the 3 iron metabolism parameters were interdependent. Hemoglobin level was positively correlated with serum iron and transferrin, and was negatively correlated with ferritin. Compared with peri-neural invasion (PNI)-negative patients, PNI-positive patients had higher serum iron (p=0.03) and ferritin levels (p=0.01). Compared with patients with the lowest quartile level of ferritin, patients with the highest quartile level of ferritin had a 2.21 (95% CI: 1.18-4.14) fold increased mortality risk in the univariate and 2.56 (95% CI: 1.10-5.96) in the multivariate Cox proportional hazards models. When stratified by TNM stages, it was only in stage III patients that serum ferritin remained statistically prognostically significant. Conclusions: Preoperative serum ferritin appeared as an independent adverse risk factor in non-metastatic colorectal cancer.