Construction Of Salt Bridges To Improve The Catalytic Activity And Thermostability Of 1,4-α-glucan Branching Enzyme

The 1,4-α-glucan branching enzyme(GBE,EC 2.4.1.18)is a glycosyltransferase belonging to glycoside hydrolase family 13(GH13)or glycoside hydrolase family 57(GH57),which catalyzes the hydrolysis of α-1,4-glycosidic linkages in starch to form short glucan chains,and the attachment of the short chains to the acceptor chains via α-1,6-glycosidic linkages to form new branches.With this unique transglycosylation,GBE has a broad application in the production of highly branched starch and slowly digestible starch.As a key enzyme for enzymatic modification of starch,the properties of GBE are crucial for the preparation of denatured starch.In a previous study,it was found that GBE from Geobacillus thermoglucosidans STB02(Gt-GBE)showed great prospects for application,however,it was found that Gt-GBE had issues such as addition of large amount of enzyme and reduction of enzyme stability during the reaction process in the application process.To overcome these drawbacks,it is crucial to improve the catalytic activity and thermostability of Gt-GBE to expand its industrial application.In this study,Gt-GBE was used to construct intramolecular salt bridges by sequence comparison and structural information analysis to select the appropriate position within the enzyme molecule to enhance the catalytic activity and thermostability of Gt-GBE.The main findings are as follows:(1)A previous study found that amino acids in the N-terminal region of Gt-GBE play an important role in its catalytic activity.Further structural analysis of this region revealed that the amino acids at position 10 and 25 in this region are spatially close to each other,and mutation of the amino acid at position 25 in Gt-GBE can significantly change the spatial structure of the N-terminal amino acid region.Thus,L25 R,L25K and L25 A mutants were constructed.The results showed that the specific activities of mutants L25 R and L25 A were 1.28 and 1.23 times higher than that of the wild-type,respectively.Structural analysis indicated that the mutation of leucine at position 25 to arginine in Gt-GBE could form an intramolecular salt bridge with glutamic at position 10 and enhance the structural rigidity of this region;the mutation of leucine at position 25 to alanine in Gt-GBE expanded the cavity structure of this region;while the mutation of leucine at position 25 to lysine in Gt-GBE disrupted the hydrophobic interactions of the region.(2)To further improve the thermostability of Gt-GBE,the amino acid sequence was analyzed to identify amino acids that may affect the thermostability of Gt-GBE.Based on the analysis of B-factor and Depth values of amino acids,we selected the amino acids with high flexibility,which are located on the surface of Gt-GBE.Subsequently,the selected amino acids and their adjacent amino acid regions were structurally analyzed,the amino acids(Gln73 and Lys137)which were suitable for constructing salt bridges were selected and intramolecular salt bridges were constructed by targeted mutagenesis.The results showed that the mutant K137 E was significantly improved in thermostability compared with the wild-type,with the half-life of mutant K137 E were 1.43 and 1.29 times higher than that of the wild-type at 60°C and 65°C,respectively.Structure analysis showed that Lys137 mutated to Asp137 in Gt-GBE could form a salt bridge with Arg140,which enhanced the local electrostatic interaction and improved the structural rigidity of the loop in which Arg140 is located.(3)To obtain mutants with higher catalytic activity and thermostability,L25 R,L25A and K137 E were selected for combined mutation,thus two combined mutants L25R/K137 E and L25A/K137 E were obtained.The results showed that compared with the wild-type,the specific activities and thermostability of the combined mutants L25R/K137 E and L25A/K137 E were improved.Subsequently,the combined mutants were modified with maltodextrin,and the application value of the combined mutants was initially explored.The results showed that after two days of storage at 4°C,the maltodextrin without Gt-GBE treatment(control)was completely turbid and the light transmittance decreased to 0.14%,the maltodextrins modified by the wild-type was slightly turbid with a light transmittance of 65.10%,while the maltodextrin modified by the combined mutants L25R/K137 E and L25A/K137 E were transparent with a light transmittance of 87.60% and 93.91%,respectively.With the extension of storage time,the maltodextrin gradually became turbid and decreased in transparency.Compared with the wildtype,the combined mutants L25R/K137 E and L25A/K137 E have higher industrial application values.