Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.
Catalysis ; Directed Molecular Evolution ; methods ; Endo-1,3(4)-beta-Glucanase ; biosynthesis ; genetics ; metabolism ; Enzyme Stability ; Hot Temperature ; Hydrogen-Ion Concentration ; Mutant Proteins ; metabolism ; Mutation ; Paecilomyces ; classification ; enzymology ; genetics ; Protein Engineering ; methods

Objective Sporadic cases occurring in mall geographic unit could lead to extreme value of incidence due to the small population bases,which would influence the analysis of actual incidence.Methods This study introduced a method of hierarchy clustering and partitioning regionalization,which integrates areas with small population into larger areas with enough population by using Geographic Information System (GIS) based on the principles of spatial continuity and geographical similarity (homogeneity test).This method was applied in spatial epidemiology by using a data set of thyroid cancer incidence in Yiwu,Zhejiang province,between 2010 and 2013.Results Thyroid cancer incidence data were more reliable and stable in the new regionalized areas.Hotspot analysis (Getis-Ord) on the incidence in new areas indicated that there was obvious case clustering in the central area of Yiwu.Conclusion This method can effectively solve the problem of small population base in small geographic units in spatial epidemiological analysis of thyroid cancer incidence and can be used for other diseases and in other areas.