ABSTRACT
The high-degree polymerization of isomaltooligosaccharide (IMO) not only effectively promotes the growth and reproduction of Bifidobacterium in the human body but also renders it resistant to rapid degradation by gastric acid and can stimulate insulin secretion. In this study, we chose the engineered strain expressed dextranase (PsDex1711) as the research model and used the AutoDock vina molecular docking technique to dock IMO4, IMO5, and IMO6 with it to obtain mutation sites, and then studied the potential effect of key amino acids in this enzyme on its hydrolysate composition and enzymatic properties by site-directed mutagenesis method. It was found that the yield of IMO4 increased significantly to 62.32% by the mutant enzyme H373A. Saturation mutation depicted that the yield of IMO4 increased to 69.81% by the mutant enzyme H373R, and its neighboring site S374R IMO4 yield was augmented to 64.31%. Analysis of the enzymatic properties of the mutant enzyme revealed that the optimum temperature of H373R decreased from 30 °C to 20 °C, and more than 70% of the enzyme activity was maintained under alkaline conditions. The double-site saturation mutation results showed that the mutant enzyme H373R/N445Y IMO4 yield increased to 68.57%. The results suggest that the 373 sites with basic non-polar amino acids, such as arginine and histidine, affect the catalytic properties of the enzyme. The findings provide an important theoretical basis for the future marketable production of IMO4 and analysis of the structure of dextranase.
Subject(s)
Amino Acids , Dextranase , Humans , Dextranase/chemistry , Dextranase/genetics , Dextranase/metabolism , Molecular Docking Simulation , Polymerization , Amino Acids/genetics , Mutagenesis, Site-DirectedABSTRACT
As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid optimization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family.
ABSTRACT
A novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), was previously discovered from Sarocladium kiliense U4520. In order to identify the factors underlying the unique substrate specificity of IMM-4IH, we endeavored to determine the amino acid sequence of the enzyme. By comparing the partial amino acid sequence of the enzyme to whole genome sequencing data of S. kiliense U4520, the IMM-4IH gene was estimated. The putative gene was expressed in Pichia pastoris, and its activity and properties were found to be consistent with those of the native enzyme. Comparing the amino acid sequence of IMM-4IH with those in the CAZy database led to classification in the glycoside hydrolase family 49 (GH49). Several amino acids important for catalysis (Asp406, Asp425, and Asp426) and substrate recognition at subsites + 1 and -3 were estimated by multiple sequence alignment analysis. These results provide important information for characterizing IMM-4IH and other GH49 enzymes.