L-Amino acid oxidases (LAAOs) are a group of FAD-containing enzymes that catalyse the oxidative deamination of L-amino acids with the production of the corresponding α-keto acid, ammonia and H2O2. Due to their strict stereoselectivity, LAAOs might be potentially employed in a variety of biotechnological processes, e.g., deracemization reactions and production of valuable chemicals or in biosensors for L-amino acids [1]. Unfortunately, the current bottleneck in the biotechnological exploitation of eukaryotic LAAOs (mainly from snake venom, the most studied enzymes of this class) is their lack of expression as recombinant proteins at suitable yields. For this reason, we focused on the overexpression of several recombinant LAAOs from microbial origin. Following an exhaustive analysis of data bank entries and literature reports, we identified different microbial LAAOs for expression studies in prokaryotic hosts. In details: i) L-aspartate oxidases from E. coli and from the thermophile archaeas Sulfolobus tokodaii and S. solfataricus, ii) LAAO from Streptococcus oligofermentas, iii) LAAO from Rhodococcus opacus, iv) and a putative LAAO from Bacillus subtilis. The heterologous expression of these proteins was evaluated using: i) different prokaryotic hosts (e.g., E. coli, Streptomyces spp); ii) different expression systems (e.g., for periplasmic expression and/or yielding chimeric proteins); iii) refolding of inclusion bodies (IB). Our results confirm the propensity of the members of this class of proteins to massively accumulate into IBs (this phenomenon probably representing an escape route to avoid cytotoxicity issues related to L-amino acid depletion and H2O2 production). Accordingly, up to now successful expression of a soluble and active protein was achieved only for LAAOs possessing a narrow substrate specificity, e.g., L-aspartate oxidase.
Recombinant L-Amino Acid oxidases: dream or reality?
MOLLA, GIANLUCA;MOTTA, PAOLO;BERINI, FRANCESCA;MARINELLI, FLAVIA;POLLEGIONI, LOREDANO;MARINELLI, FLAVIA
2014-01-01
Abstract
L-Amino acid oxidases (LAAOs) are a group of FAD-containing enzymes that catalyse the oxidative deamination of L-amino acids with the production of the corresponding α-keto acid, ammonia and H2O2. Due to their strict stereoselectivity, LAAOs might be potentially employed in a variety of biotechnological processes, e.g., deracemization reactions and production of valuable chemicals or in biosensors for L-amino acids [1]. Unfortunately, the current bottleneck in the biotechnological exploitation of eukaryotic LAAOs (mainly from snake venom, the most studied enzymes of this class) is their lack of expression as recombinant proteins at suitable yields. For this reason, we focused on the overexpression of several recombinant LAAOs from microbial origin. Following an exhaustive analysis of data bank entries and literature reports, we identified different microbial LAAOs for expression studies in prokaryotic hosts. In details: i) L-aspartate oxidases from E. coli and from the thermophile archaeas Sulfolobus tokodaii and S. solfataricus, ii) LAAO from Streptococcus oligofermentas, iii) LAAO from Rhodococcus opacus, iv) and a putative LAAO from Bacillus subtilis. The heterologous expression of these proteins was evaluated using: i) different prokaryotic hosts (e.g., E. coli, Streptomyces spp); ii) different expression systems (e.g., for periplasmic expression and/or yielding chimeric proteins); iii) refolding of inclusion bodies (IB). Our results confirm the propensity of the members of this class of proteins to massively accumulate into IBs (this phenomenon probably representing an escape route to avoid cytotoxicity issues related to L-amino acid depletion and H2O2 production). Accordingly, up to now successful expression of a soluble and active protein was achieved only for LAAOs possessing a narrow substrate specificity, e.g., L-aspartate oxidase.
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