A bacterial strain having 3-deoxyglucosone-metabolizing enzyme was selected from 31 marine bacterial stains. The conditions for enzyme production of the strain was examined. The optimal tempreture, initial pH. and cultivate time for enzyme formation were 28℃, pH7.8~8.0, and 96 hours respectively. Composition of the suitable medium was as following (%): Fish peptone 1.0, Sucrose 0.3, Yeast extract 0.2, NaCI 5.0.3-deoxyglucosone can induce formation of enzyme.

Functional improvement to one component of the cellulase, endo-beta-1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo-beta-1,4-glucanase (NtEG PDB id = 1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56IIe showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.
Amino Acids ; genetics ; Animals ; Cellulase ; chemistry ; genetics ; metabolism ; Isoptera ; enzymology ; Mutagenesis, Site-Directed ; Mutation

Objective:To investigate the change of peptidylarginine deiminase 4 (PAD4) expression in the neutrophils of peripheral blood of patients with anti-neutrophil cytoplasmic myeloperoxidase antibody-associated vasculitis (MPO-AAV), and to analyze the relationship between the change and disease activity.Methods:Thirty-nine untreated patients with active MPO-AAV (patient group) and thirty-nine healthy volunteers (control group) were enrolled into this study. The PAD4 expressed on neutrophils was detected by flow cytometry (FCM). The serum neutrophil extracellular traps (NETs), fragments from the activated complement C5 (C5a) and anti-neutrophil cytoplasmic myeloperoxidase antibody (MPO-ANCA) were measured by enzyme-linked immuno sorbent assay (ELISA). Their disease activity was evaluated by Birmingham vasculitis activity score (BVAS). All the detected results were compared between the 2 groups by t test, Spearman correlation analysis and multivariate linear regression analysis were employed to analyze the relationship between BVAS and the Lab test results in patient group. Results:The proportion of neutrophil expressing PAD4, the mean fluorescence intensity (MIF) of PAD4, the levels of NETs and C5a in patient group were significantly higher than those in the control group [(71±11)% vs (26±6)%, t=22.456, P<0.01; (33±4) vs (14±4), t=18.668, P<0.01; (0.62±0.22) vs (0.26±0.15), t=8.466, P<0.01 and (4.6±1.0) vs (2.9±1.0), t=7.697, P<0.01, respectively]. In patient group, BVAS was positively associated with the proportion of PAD4 + neutrophil, MFI of PAD4, the serum level of NETs, C5a and MPO-ANCA ( r=0.843, P<0.01; r=0.821, P<0.01; r=0.411 1, P<0.01; r=0.613, P<0.01 and P=0.790, P<0.01, respectively), however, the results of multiple linear regression analysis showed only the percentage of PAD4 + neutrophils and the level of MPO-ANCA were independent influencing factors on BVAS ( β=0.324 6, P<0.01 and β=0.796, P<0.01, respectively). Conclusion:The percentage of neutrophils expressed PAD4 in the peripheral blood of patient with active MPO-AAV is significantly increased, and it is an independent factor affecting the disease activity. Intervention on this expression might be a potential new pathway for MPO-AAV treatment.