A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178

Abstract

l-tert-leucine and its derivatives are useful as pharmaceutical active ingredients, in which leucine dehydrogenase (LeuDH) is the key enzyme in their enzymatic conversions. In the present study, a novel cold-adapted LeuDH, psleudh, was cloned from psychrotrophic bacteria Pseudoalteromonas sp. ANT178, which was isolated from Antarctic sea-ice. Bioinformatics analysis of the gene psleudh showed that the gene was 1209 bp in length and coded for a 42.6 kDa protein containing 402 amino acids. PsLeuDH had conserved Phe binding site and NAD+ binding site, and belonged to a member of the Glu/Leu/Phe/Val dehydrogenase family. Homology modeling analysis results suggested that PsLeuDH exhibited more glycine residues, reduced proline residues, and arginine residues, which might be responsible for its catalytic efficiency at low temperature. The recombinant PsLeuDH (rPsLeuDH) was purified a major band with the high specific activity of 275.13 U/mg using a Ni-NTA affinity chromatography. The optimum temperature and pH for rPsLeuDH activity were 30 °C and pH 9.0, respectively. Importantly, rPsLeuDH retained at least 40% of its maximum activity even at 0 °C. Moreover, the activity of rPsLeuDH was the highest in the presence of 2.0 M NaCl. Substrate specificity and kinetic studies of rPsLeuDH demonstrated that l-leucine was the most suitable substrate, and the catalytic activity at low temperatures was ensured by maintaining a high kcat value. The results of the current study would provide insight into Antarctic sea-ice bacterium LeuDH, and the unique properties of rPsLeuDH make it a promising candidate as a biocatalyst in medical and pharmaceutical industries.

Advances in cold-adapted enzymes derived from microorganisms

“…Cold-adapted enzyme amino acid sequence analysis shows that the number of proline residues in cold-adapted enzymes is much lower than that in mesophilic and thermophilic enzymes (Wang Y. et al, 2018;Hou et al, 2019). Proline's α-carbon atom is connected to its five-membered ring structure side chain group, which makes it difficult for the main chain carbon atom to rotate This limits the rotation of the carbon main chain during the formation of a peptide chain (Figure 7B), increases the rigidity of the peptide chain, and reduces local flexibility of the protein, but to a certain extent, it improves the stability of a protein, namely the 'proline law' (Watanabe et al, 1996).…”

“…However, cold-adapted enzyme resource development has found some cold-adapted α-amylases that possess a degree of tolerance to metal and salt ions. For instance, Wang Y. et al (2018) and Wang X. et al (2018) analyzed and characterized a cold-adapted α-amylase from an Antarctic bacterium and found the enzyme to be extremely salt-tolerant. It also possesses a degree of tolerance to other metal ions, such as potassium, calcium, and manganese, and to solvents, including sodium dodecyl sulfate (SDS), and dimethyl sulfoxide (DMSO).…”

Bioactive Molecules from Extreme Environments

“…A cold-adapted leucine dehydrogenase (EC 1.4.1.9), a NAD + -dependent oxidoreductase, with unique substrate specificity was cloned from the Antarctic marine psychrotrophic bacterium Pseudoalteromonas sp. ANT178 and was shown to retain 40% of its maximal activity at 0 • C. Being the key enzyme in the enzymatic conversion of L-leucine and other branched chain L-amino acids in the corresponding α-keto acid, it was suggested as biocatalyst in the pharmaceutical industry [157].…”

“…GWS-BW-H8hM For instance, in Mediterranean DHABs transcripts related to Streptomyces have been identified, thus representing an important source of bioactive natural products with clinical or pharmaceutical applications [71,155]. Additionally, Pseudoalteromonas flavipulchra recently isolated from the Nereus halocline shows great antimicrobial activity which is associated with the different metabolites and/or enzymes that this species can produce [84,156,157].…”

Enzymes from Marine Polar Regions and Their Biotechnological Applications

“…ANT178 and was shown to retain 40% of its maximal activity at 0 °C. Being the key enzyme in the enzymatic conversion of L-leucine and other branched chain L-amino acids in the corresponding α-keto acid, it was suggested as biocatalyst in the pharmaceutical industry [157].…”