Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

Abstract

The ascomycin-producer strain Streptomyces ascomycinicus has been proven to be an extracellular poly(R)-3-hydroxybutyrate (PHB) degrader. The fkbU gene, encoding a PHB depolymerase (PhaZSa), has been cloned in E. coli and Rhodococcus sp. T104 strains for gene expression. Gram-positive host Rhodococcus sp. T104 was able to produce and secrete to the extracellular medium an active protein form. PhaZSa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemically as well as structurally characterized. The enzyme was found to be a monomer with a molecular mass of 48.4 kDa, and displayed highest activity at 45°C and pH 6, thus being the first PHB depolymerase from a gram-positive bacterium presenting an acidic pH optimum. The PHB depolymerase activity of PhaZSa was increased in the presence of divalent cations due to non-essential activation, and also in the presence of methyl-β-cyclodextrin and PEG 3350. Protein structure was analyzed, revealing a globular shape with an alpha-beta hydrolase fold. The amino acids comprising the catalytic triad, Ser131-Asp209-His269, were identified by multiple sequence alignment, chemical modification of amino acids and site-directed mutagenesis. These structural results supported the proposal of a three-dimensional model for this depolymerase. PhaZSa was able to degrade PHB, but also demonstrated its ability to degrade films made of PHB, PHBV copolymers and a blend of PHB and starch (7∶3 proportion wt/wt). The features shown by PhaZSa make it an interesting candidate for industrial applications involving PHB degradation.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs

“…Vertical blue lines represent gaps in the complete sequence enabling the expression of heterologous genes in Rhodococcus sp. T104, as previously demonstrated (García- Hidalgo et al , 2013. Remarkably, the abilitiy of Rhodococcus sp.…”

Poly-𝛽-hydroxy butyrate Depolymerase from Streptomyces lydicus MM10, Isolated from Wastewater Sample

“…Similar results were obtained by García-Hidalgo et al (2013). Further researches are required for exploiting the interesting characters of depolymerase enzymes and their roles in PHB degradation.…”

“…Optimum temperature and pH for enzyme activity depend on strain type. The first PHB depolymerase from a gram-positive were from S. ascomycinicus and the enzyme was found to be a monomer with a molecular mass of 48.4 kDa, with the highest activity at 45°C and pH 6 and the activity was increased in the presence of divalent cations (García-Hidalgo et al, 2013). The thermophilic Streptomyces sp.…”

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters

“…Over the past few decades, the identification of PHA depolymerases has received large attention (Knoll et al, 2009;García-Hidalgo et al, 2013;Martínez-Tobón et al, 2018;Sayyed et al, 2019). Despite the extant sequence variability amongst these enzymes, all intra-and extracellular PHA depolymerases share a common α/β-hydrolase fold and a catalytic triad (Knoll et al, 2009).…”