3 reference(s) found. Listing paper details in reverse chronological order. We are grateful to Keith Bradnam for improvment of this script
Title: Characterization of Polymer Degrading Lipases, LIP1 and LIP2 From Pseudomonas chlororaphis PA23 Mohanan N, Wong CH, Budisa N, Levin DB Ref: Front Bioeng Biotechnol, 10:854298, 2022 : PubMed
The outstanding metabolic and bioprotective properties of the bacterial genus Pseudomonas make these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacterium Pseudomonas chlororaphis PA23 and subsequently performed cloning and expression in Escherichia coli. The lip1 gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas the lip2 consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant alpha/beta-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyanion pocket, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with an oxyanion pocket of GGX-type, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. Our three-dimensional models of LIP1 and LIP2 complexed with a 3-hydroxyoctanoate dimer revealed the core alpha/beta hydrolase-type domain with an exposed substrate binding pocket in LIP1 and an active-site capped with a closing lid domain in LIP2. The recombinant LIP1 was optimally active at 45 degreesC and pH 9.0, and the activity improved in the presence of Ca(2+). LIP2 exhibited maximum activity at 40 degreesC and pH 8.0, and was unaffected by Ca(2+). Despite different properties, the enzymes exhibited broadsubstrate specificity and were able to hydrolyze short chain length and medium chain length polyhydroxyalkanoates (PHAs), polylactic acid (PLA), and para-nitrophenyl (pNP) alkanoates. Gel Permeation Chromatography (GPC) analysis showed a decrease in the molecular weight of the polymers after incubation with LIP1 and LIP2. The enzymes also manifested some polymer-degrading activity on petroleum-based polymers such as poly(sigma-caprolactone) (PCL) and polyethylene succinate (PES), suggesting that these enzymes could be useful for biodegradation of synthetic polyester plastics. The study will be the first report of the complete characterization of intracellular lipases from bacterial and/or Pseudomonas species. The lipases, LIP1 and LIP2 are different from other bacterial lipases/esterases in having broad substrate specificity for polyesters.
Title: Biodegradation mechanism of polycaprolactone by a novel esterase MGS0156: a QM/MM approach Feng S, Yue Y, Chen J, Zhou J, Li Y, Zhang Q Ref: Environ Sci Process Impacts, 22:2332, 2020 : PubMed
Nowadays micro-plastic pollution has become one of the most serious global environmental problems. A potential strategy in managing micro-plastic waste is enzyme-catalyzed degradation. MGS0156 is a hydrolase screened from environmental metagenomes, which can efficiently degrade commercial plastics such as polycaprolactone, polylactide, etc. Here a combined molecular dynamics, molecular mechanics Poisson-Boltzmann surface area, and quantum mechanics/molecular mechanism method was used to reveal the enzymatic depolymerization mechanism. By systematically analyzing the binding processes of nine oligomers (from a monomer to tetramer), we found that longer oligomers have relatively stronger binding energy. The degradation process involves two concerted elementary steps: triad-assisted nucleophilic attack and C-O bond cleavage. C-O bond cleavage is the rate determining step with an average barrier of 15.7 kcal mol-1, which is consistent with the experimentally determined kcat (1101 s-1, corresponds to 13.3 kcal mol-1). The electrostatic influence analysis of twenty amino acids highlights His231 and Asp237 as potential mutation targets for designing more efficient MGS0156 mutants.
Title: Purification, characterization, and gene cloning of an Aspergillus fumigatus polyhydroxybutyrate depolymerase used for degradation of polyhydroxybutyrate, polyethylene succinate, and polybutylene succinate Jung HW, Yang MK, Su RC Ref: Polymer Degradation and Stability, 154:154, 2018 : PubMed
Aspergillus fumigatus strain 76T-3 formed clear zones on agar plates containing emulsified polyhydroxybutyrate (PHB), polyethylene succinate (PES), polybutylene succinate (PBS), polycaprolactone (PCL), or polylactide (PLA). The strain grew well at 40 C in Sabouraud Dextrose Broth. Solution-casted PHB films were almost completely degraded after incubation with 76T-3 at 45 C for 17 h. An extracellular polyester-degrading enzyme was purified from the supernatant of 76T-3 cultures in basal medium containing PHB as the sole carbon source. Zymography results portrayed that the purified enzyme degraded PHB, PES, and PBS but not PCL or PLA. The amino acid sequence obtained from LC-MS/MS identified this enzyme to be a PHB depolymerase with a molecular mass of 57 kDa. The optimal reaction condition for the enzyme was pH 6.4 at 55 C. The recombinant PHB depolymerase (rPhaZ) expressed in E. coli showed the enzyme can act on PHB only and not on PES or PBS.
