A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

Abstract

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

“…Large-scale (4 L fermentative) production and Ni-NTA enrichment of BLC23O Shake ask production of BLC23O from Echerichia coli has been previously reported (Adewale et al, 2021). Here production of BLC23O was scaled up to a 4 L fermentative format.…”

“…An optimized version of the BLC23O coding region was cloned into the pET28B + vector such that the expressed fusion protein included an N-terminal 6 x His tag as previously described (Adewale et al, 2021). Echerichia coli BL21 cells containing the expression vector encoding the BLC23O gene was used to inoculate an overnight culture containing 50 mL of Terri c Broth (TB) medium (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) with 50 µg/mL kanamycin.…”

“…Thus, reducing phenol content (tannic and non-tannic) in pulse products remains an important goal for increased nutritional and economic value. (Adewale et al, 2021). This enzyme was found to have unusually broad substrate speci city and good thermostability, traits proposed to be associated with its atypical monomeric structure.…”

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

“…One potential candidate for cell-free phenolic degradation of pulse meal is a Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O; (Adewale et al 2021 )). B. ligniniphilus L1 is a halotolerant and alkaliphilic bacterium isolated from sediments from the South China Sea, known to use poly-phenolic lignin as its sole carbon source.…”

“…Catechol 2,3-dioxygenases elicit proximal extradiol cleavage. Three catechol 2,3-dioxygenases encoded by B. ligniniphilus L1 have been identified and the shortest one, a protein of 283 amino acids (BLC23O, NCBI accession WP_017726464.1) with a calculated molecular mass of approximately 32 kDa, was recently characterized (Adewale et al 2021 ). This enzyme was found to have unusually broad substrate specificity and good thermostability, traits proposed to be associated with its atypical monomeric structure.…”