Modification of the Loop Region Near the Substrate Tunnel to Alter the Hydrolytic Process of Dextranase.

Dextranases are hydrolases that exclusively catalyze the disruption of α-1,6 glycosidic bonds. A series of variant enzymes were obtained by comparing the sequences of dextranases from different sources and introducing sequence substitutions. A correlation was found between the number of amino acids in the 397-401 region and the hydrolytic process. When there were no more than 5 amino acids in the 397-401 region, the enzyme first hydrolyzed the dextran T70 to a low molecular weight dextran with a molecular weight of about 5000, then IMOs1 appeared in the system if the degradation continued, showing a clear sequential relationship. And when there are more than 5 amino acids in the 397-401 region, IMOs were produced at the beginning of hydrolysis and continue to increase throughout the hydrolytic process. At the same time, we investigated the enzymatic properties of the variants and found that the hydrolytic rate of A-Ca was 11 times higher than that of the original enzyme. The proportion of IMOs produced by A-Ca was 80.68%, which was nearly10% higher than the original enzyme, providing a new enzyme for the industrial preparation of IMOs.

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 %).