(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Actinobacteria [phylum]: NE > Actinobacteria [class]: NE > Corynebacteriales: NE > Nocardiaceae: NE > Rhodococcus: NE > Rhodococcus fascians: NE > Rhodococcus fascians D188: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MKRRLIAYSIAALAISATAVALPTGVASAAPCSDVDVSFARGTGELPGLG ITGTPFVNSVKSQLSDRSVSTYAVNYAADFTQASAGPGSRDLVAHLNSVA ASCPSTKFVIGGYSQGATVVTNAVGLRTPSSFTGAVIPAAIADRIEAVVV FGNPFGLTGRKIETASSTYGSRTNSFCNFGDPVCQIGGFNTFAHLTYGTN GSTTQGASFAAAQVRS
References
Title: Functional production, characterization, and immobilization of a cold-adapted cutinase from Antarctic Rhodococcus sp Won SJ, Yim JH, Kim HK Ref: Protein Expr Purif, :106077, 2022 : PubMed
A lipolytic enzyme (Rcut) was discovered from the Rhodococcusstrain (RosL12) isolated from the Antarctic Ross Sea. The corresponding gene composed of 651 bases encoding 216 amino acids. It was found to be a cutinase gene through BLAST search. Rcut has a signal sequence consisting of 29 amino acids. An active Rcut was produced after the intact gene containing the signal sequence was transformed into Escherichia coli Rosetta-gami 2 (DE3) pLysS. Rcut was purified through a nickel-nitrilotriacetic acid purification system and a carboxymethyl Sepharose column chromatography. Its specific activity was 2190 U/mg. Rcut showed the highest activity at 40 degreesC and had a low activation energy of 3.16 kcal/mol. This means that it is a typical cold-adapted enzyme. Rcut showed high activity towards medium chain fatty acids (C(4)-C(10)). Rcut degraded polycaprolactone and polyethylene terephthalate, suggesting that it could be used for decomposition of synthetic plastics causing environmental pollution. Rcut was immobilized on methacrylate-divinyl benzene bead. This immobilized Rcut (immRcut) showed higher thermal stability than the free enzyme. ImmRcut performed transesterification of various esters and ethanol in a non-polar solvent, suggesting that it could be used for the synthesis of industrially useful ester compounds.
Title: Synthesis of Short-Chain Alkyl Butyrate through Esterification Reaction Using Immobilized Rhodococcus Cutinase and Analysis of Substrate Specificity through Molecular Docking Won SJ, Yim JH, Kim HK Ref: J Microbiol Biotechnol, 33:1, 2022 : PubMed
Alkyl butyrate with fruity flavor is known as an important additive in the food industry. We synthesized various alkyl butyrates from various fatty alcohol and butyric acid using immobilized Rhodococcus cutinase (Rcut). Esterification reaction was performed in a non-aqueous system including heptane, isooctane, hexane, and cyclohexane. As a result of performing the alkyl butyrate synthesis reaction using alcohols of various chain lengths, it was found that the preference for the alcohol substrate had the following order: C6 > C4 > C8 > C10 > C2. Through molecular docking analysis, it was found that the greater the hydrophobicity of alcohol, the higher the accessibility to the active site of the enzyme. However, since the number of torsions increased as the chain length increased, it became difficult for the hydroxyl oxygen of the alcohol to access the gamma O of serine at the enzyme active site. These molecular docking results were consistent with substrate preference results of the Rcut enzyme. The Rcut maintained the synthesis efficiency at least for 5 days in isooctane solvent. We synthesized as much as 452 mM butyl butyrate by adding 100 mM substrate daily for 5 days and performing the reaction. These results show that Rcut is an efficient enzyme for producing alkyl butyrate used in the food industry.
Title: Characterization of a poly(butylene adipate- co -terephthalate) hydrolase from the mesophilic actinobacteria Rhodococcus fascians Soulenthone P, Tachibana Y, Suzuki M, Mizuno T, Ohta Y, Kasuya KI Ref: Polymer Degradation and Stability, 154:109481, 2021 : PubMed
Poly(butylene adipate- co -terephthalate) (PBAT) possesses excellent film-forming ability and biodegrad- ability. Therefore, it is considered to be a promising mulching film material that eliminates the need for recovery. In the applications that require PBAT degradation in the field after use, it is important to un- derstand the biodegradation mechanism at moderate temperatures. We have previously isolated from the soil the mesophilic actinobacteria Rhodococcus fascians NKCM2511 that biodegraded PBAT under moderate temperature conditions (20-30 C). In this study, to clarify the mechanism of PBAT degradation by the strain NKCM2511, a DNA fragment carrying the gene pbath Rf responsible for the PBAT degradation activity was cloned. The gene encoded a 216-amino-acid-long protein designated as PBATH Rf . Homology modeling revealed that PBATH Rf belongs to the alpha/ betahydrolase fold family, lacking the lid domain covering the active site. PBATH Rf degraded PBAT film at 30 C at the rate of 0.10 +/- 0.03 mg/cm 2 /d and was capable of degrad- ing several other aliphatic polyester films. Liquid chromatography revealed that PBATH Rf preferentially cleaved the ester bond between 1,4-butanediol and adipic acid rather than that between 1,4-butanediol and terephthalic acid (T). This characteristic of PBATH Rf may explain the low degradation rate of the aliphatic - aromatic copolyester PBAT, compared to the rate of degradation of aliphatic polyesters without T. In addition, liquid chromatography showed that PBATH Rf released T, mono(2-hydroxyethyl) terephthalic acid, and bis(2-hydroxybutyl) terephthalate from an amorphous poly(ethylene terephthalate) (PET) film. However, no significant change in the PET film surface after the treatment with PBATH Rf was found by scanning electron microscopy. This is the first report of an enzyme from the mesophilic actinobacteria Rhodococcus fascians that can hydrolyze various polyesters, including PBAT, and catalyze hydrolysis on the surface of an amorphous PET film. This study also provides insight into the biodegradation mechanism of PBAT in the actual field as it describes an enzyme from a naturally occurring organism that acts in the medium temperature range.
