Improving the thermostability of GH49 dextranase AoDex by site-directed mutagenesis

Abstract

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.

The effects of the chemical modification on immobilized lipase features are affected by the enzyme crowding in the support

“…5 As industrial catalysts, enzymes should be heterogeneous biocatalysts (to facilitate enzyme recovery and reaction control), stable, active, selective, and specific for the applied targeted substrate and reaction under industrially relevant conditions. [1][2][3][4] Fortunately, there are many tools that enable to bridge this gap, such as metagenomics, that enables to utilize most biodiversity, [6][7][8][9] directed evolution, that permits to mimic natural selection but focusing on the enzyme and the feature that the researcher has selected in an accelerated way, [10][11][12] directed mutagenesis, [13][14][15][16] that enables to build new enzymes, even enzymes bearing several different enzymes 17 (e.g., plurizymes), [18][19][20][21] and some physicochemical tools. Among the last ones, chemical modification of the enzyme surface can reach diverse objectives, such as altering the enzyme surface features, the mobility of a specific section of the protein or introducing intra or intermolecular crosslinkings.…”