Abstract
The α-glucosidases play indispensable roles in the metabolic mechanism of organism, prevention, and treatment of the disease, and sugar hydrolysis, and are widely used in chemical synthesis, clinical diagnosis, and other fields. However, improving their catalytic efficiency and production to meet commercial demand remains a huge challenge. Here we detected a novel GH13 family α-glucosidase, QsGH13, from the deep-sea bacterium Qipengyuania seohaensis sp. SW-135. QsGH13 is highly substrate specific and only hydrolyzes sugars containing alpha-1,4 glucoside bonds. For example, its enzymatic activity for p-nitrophenyl-α-D-glucopyranoside was 25.41 U/mg, and the Km value was 0.2952 ± 0.0322 mM. The biochemical results showed that the optimum temperature of QsGH13 is 45°C, the optimum pH is 10.0, and it has excellent biological characteristics such as alkali resistance and salt resistance. The crystal structure of QsGH13 was resolved with a resolution of 2.2 Å, where QsGH13 is composed of a typical TIM barrel catalytic domain A, a loop-rich domain B, and a conserved domain C. QsGH13 crystal belonged to the monoclinic space group P212121, with unit-cell parameters a = 58.816 Å, b = 129.920 Å, c = 161.307 Å, α = γ = β = 90°, which contains two monomers per asymmetric unit. The β → α loop 4 of QsGH13 was located above catalytic pocket. Typical catalytic triad residues Glu202, Asp266, and Glu329 were found in QsGH13. The biochemical properties and structural analysis of QsGH13 have greatly improved our understanding of the catalytic mechanism of GH13 family. This study provides new ideas to broaden the application of α-glucosidase in alcohol fermentation, glycolysis, and other industries.
Highlights
The CAZy database1 classifies proteins into glycoside hydrolases (GHs), glycosyl transferases, polysaccharide lyases, carbohydrate esterases, auxiliary activities, and carbohydrate-binding modules based on amino acid sequence similarity and spatial structure of their catalytic structural domains (Henrissat, 1991; Cantarel et al, 2009)
Phylogenetic tree sequence analysis based on NCBI, PDB, and CAZy databases showed that the protein qsgh13 encoding α-glucosidase (QsGH13) belongs to the GH13 family (Supplementary Figure 1), and predicted that the protein QsGH13 had α-glucosidase characteristics
After the His6-SUMO tag was removed by Ulp1 enzyme, the protein QsGH13 was purified by gel-filtration chromatography to 95% homogeneity
Summary
The CAZy database classifies proteins into glycoside hydrolases (GHs), glycosyl transferases, polysaccharide lyases, carbohydrate esterases, auxiliary activities, and carbohydrate-binding modules based on amino acid sequence similarity and spatial structure of their catalytic structural domains (Henrissat, 1991; Cantarel et al, 2009). The enzymes of the GH13 family catalyze reactions mainly through a double-displacement mechanism, in which catalytic nucleophilic (Asp) and acid/base catalyst (Glu) play an essential role (McCarter and Withers, 1994). The α-glucosidases (EC:3.2.1.20) are members of GHs and are widely distributed in all organisms that are specific mainly for the exohydrolysis of α-1,4-glycosidic linkages at the nonreducing end of polysaccharides, such as maltooligosaccharides, and release α-D-glucose. It combines free glucose residues with the α-1,4-glycosidic linkages in oligosaccharides to form α-1,6-glycosidic linkages, and obtain non-fermentative oligosaccharides (McCarter and Withers, 1994; Hondoh et al, 2008). It is urgent to find novel α-glucosidase to improve glucose production and catalytic efficiency
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