Fusion enzyme design based on the "channelization" cascade theory and homogenous dextran product improvement

Homogeneous low molecular weight dextran can be used to improve microcirculation and expand blood volume. However, the synthesis and separation of low molecular weight dextran are chemically difficult and environmentally unfriendly. Here, a one-step strategy for the synthesis of homogeneous low molecular weight dextran was developed. Dextransucrase and dextranase were fused by the addition of different length linker peptides. An artificial bifunctional enzyme was created to directly convert sucrose into low molecular weight dextran (13,050 Da), and the related substrate channel mechanism was found. The substrate channel adaptability was studied by changing the length of the linker and its corresponding product behavior. Compared with the mixture of two free enzymes, the residence lag time demonstrates the degree of substrate channelization of a series of fusion enzymes. And found that the highest channelization degree is not equal to produce homogenous dextran. Whereas a fusion enzyme with the appropriate linker (the one with the best substrate channel adaptation) will produce dextran with a homogeneous molecular weight. By studying the temperature dynamics of the fusion enzyme to adjust the two-stage catalytic efficiency of the fusion enzyme, we have increased the yield of low molecular weight homogeneous dextran (Yield of 62 %).