US Patent 11142752 Pullulanase mutant

The disclosure herein relates to a method for reducing the inhibitory effect of a cyclodextrin on a pullulanase and belongs to the technical field of gene engineering, enzyme engineering or food science. The method of the disclosure prepares a pullulanase mutant by reasonably mutating the key amino acid of the pullulanase interactive with the cyclodextrin to reduce the inhibitory effect of the cyclodextrin on the pullulanase, thereby improving the hydrolysis activity of the pullulanase. The disclosure finds the interactive sites of the pullulanase and the cyclodextrin based on analysis of crystal structures of enzymes and inhibitors and sequence comparison of enzymes from different sources, and utilizes site-directed mutation to obtain the pullulanase mutant having reduced sensibility to the cyclodextrin, thereby improving the utilization ratio of the starch raw material and the yield of the cyclodextrin.