Thermostability and catalytic ability enhancements of 1,4-α-glucan branching enzyme by introducing salt bridges at flexible amino acid sites

Abstract

The 1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18), is reported to catalyze the formation of α-1,6 branching points in carbohydrates, which has ability of changing the structure of starch by adjusting the points and frequency of branch points in starch. In practical uses, GBEs are expected to be used at relatively high temperature because of high temperature requirement of starch gelatinization process. However, the present GBE does not meet the requirements, which limits the application of GBEs in starch modification industry. In order to enhance the thermostability of GBE, we combined the values of B-factor and accessible surface area, as well as the opportunity of building potential salt bridges in this enzyme. As a result, amino acid sites 73 and 137 in GBE from Geobacillus thermoglucosidans STB02 were selected in our research. The results show that substitution of Lys with Glu at amino acid 137 site resulted in an approximately 36.6 % enhancement in half-time; replacement of Gln with Asp at this site resulted in a 26 % improvement in thermostability. In addition to thermostability, catalytic ability is another factor to be concerned in our study. We constructed combined mutants with high catalytic efficiency and high thermostability. Compared with wild type, the resulting mutants have advantages in maltodextrin modification, of which product exhibited stronger stability than unmodified maltodextrin. Our study provides a new approach to increase thermostability of present GBE and create mutants with high thermostability and catalytic ability.

Keywords: 1,4-α-Glucan branching enzyme; B-factor; Residue depth value.