Improvement Of Thermostability Of Bacillus Amyloliquefaciens Bs5582 β-1,3-1,4-glucanase Through Vitro Evolution

Enzymes are efficient and specific biocatalysts widely used in food industries.β-1,3-1,4-Glucan is a polymer composed of glucose monomers linked byβ-1,3 andβ-1,4-glycosidic bonds in an irregular fashion. This polymer is abundant in the endosperm cell walls of barley, rye, sorghum, rice, and wheat Barleyβ-glucan is water-soluble and its water solution often has a high viscosity.β-glucan will be released during mashing, which may cause severe problems to beer brewing, such as the reduced yield of malt extracts, the decreased filtration rate, and the appearance of gelatinous precipitates in finished beer. In vitro evolution methods are often used to modify protein with improved characteristics. In this study,we have developed a directed evolution protocol to enhance the thermostability of theβ-1,3-1,4-glucanase.The thermostability of enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, half-life value and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01,2-JF-02 and 2-JF-03 were increased by 2.2℃,5.5℃and 3.5℃,respectively,the half-life values(t1/2,60℃) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4min,13min and 17min, respectively, the Vmax of mutant enzymes were decresed by 8.3%,2.6% and 10.6%,respectively,while,Km of mutant enzymes were nearly unchanged.Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S,G213R),2-JF-02 (C86R,S115I,N150G) and 2-JF-03 (E156V,K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of theβ-1, 3-1, 4-glucanase.This study provide some intresting results of the structural basis of the thermostability ofβ-1, 3-1, 4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.This study improved the thermostability ofβ-1, 3-1, 4-glucanase by directed-evoluation provide new data on the structural basis of the thermostability ofβ-1, 3-1, 4-glucanase, and provide a basis for the design of new, improved forms of the enzyme for future industrial use. It has been shown that the packing interactions between residues in the interior of the protein, hydrogen bonding and hydrophobic effect are dominant factors that define protein stability. It is interesting that three of these mutations are arginine substitutions. Shallowly buried argnines are likely to influence the overall electrostatic potential of the protein surface. Several studies indicate that there is a correlation between protein stability and the number of arginines on the protein surface. We also found that five of these mutations are on the surface of the enzyme that shows an obvious preference for surface mutations[1]. On one hand , surface residues forming proportionally fewer interactions than'internal'residues, and therefore being less likely to cause detrimental effects that offset gains by mutation; on the other hand, protein surfaces have been suggested to be important for thermostability of proteins showing irreversible unfolding. These variants, therefore, have improved thermostability and are ideal candidates for DNA shuffling experiments to produce a robustβ-1, 3-1, 4-glucanase for industrial application.