Affiliation:
1. Department of Biotechnology College of Life Sciences Ritsumeikan University 1-1-1 Noji-higashi Kusatsu Shiga 525-8577 Japan
2. Department of Electrical and Computer Engineering Toyama Prefectural University 5180 Kurokawa Imizu Toyama 939-0398 Japan
3. Current address: Department of Applied Chemistry and Bioscience Chitose Institute of Science and Technology 758-65 Bibi Chitose Hokkaido 066-8655 Japan.
Abstract
AbstractSuccessful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L‐amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6‐point and 58‐point mutants showing a 2.64‐ and 4.22‐fold increase, respectively, compared with WT‐LAO. Ultimately, using recombinant LAO in the biotransformation of α‐keto acids indicates its great potential as a biocatalyst in industrial production.