Open AccessRecombinant expression of an l ‐amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation
Author(s) -
Heß Marc Christian,
Bloess Svenja,
Risse Joe Max,
Friehs Karl,
Fischer von Mollard Gabriele
Publication year - 2020
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.1112
Subject(s) - pichia pastoris , biochemistry , fermentation , pichia , yeast , chemistry , amino acid , oxidative deamination , enzyme , hydrogen peroxide , recombinant dna , biology , gene
l ‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l ‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His‐tag and compare this with the recently characterized 6His‐ hc LAAO4 expressed in E . coli . The expression of the enzyme with an ER‐signal sequence in P . pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P . pastoris compared to E . coli . Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, K m, and v max values, hc LAAO4 showed very few differences when produced in these two expression systems. A higher yield of hc LAAO4 could be obtained by fermentation.