Insights into the biodegradation of polyhydroxyalkanoates by the tropical marine isolate, Nocardiopsis dassonvillei NCIM 5124

Abstract

In the current study, the ability of an indigenous marine Actinomycete Nocardiopsis dassonvillei (NCIM 5124) to degrade poly(3-hydroxybutyrate)-PHB was examined. From the whole genome sequencing data of the organism, information regarding the PHB depolymerase gene and amino acid sequence (Accession number: MCK9871921.1) was retrieved. In silico studies indicated the presence of a signal peptide characteristic of extracellular enzymes. ProtParam tool predicted that the protein had a molecular mass of 42.46 kDa with an isoelectric point of 4.51. Aliphatic and instability index values suggested that the protein was stable and the observed GARVY value indicated its hydrophilic nature. 3D structure prediction and multiple sequence alignments revealed the presence of Type I catalytic domain [including the oxyanion histidine towards the N terminal, the catalytic triad with serine (as a part of GLSAG pentapeptide), aspartate and histidine], substrate binding and linker domain. The organism was able to grow on PHB in solid and liquid media and effectively degrade it. Maximum enzyme activity (1.8 U/mL/min) was observed after 5 d of incubation in Bushnell Hass Medium containing 0.1% PHB, 1.5% sodium chloride, at 30 °C, pH 7.5 with agitation at 130 rpm. Application of the organism in disintegrating films of PHB and its copolymers was successfully demonstrated on the basis of weight loss and scanning electron microscope analysis. To the best of our knowledge, this is the first report on production of PHB depolymerase with high efficiency by N. dassonvillei, an organism that holds promise in degrading PHB-derived waste material.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-024-04079-3.

Keywords: Biodegradation; Extracellular enzyme; Nocardiopsis dassonvillei; Polyhydroxyalkanoates; Polyhydroxybutyrate depolymerase.

Fig. 1

In silico studies on PHB depolymerase derived from N. dassonvillei NCIM 5124 a predicted structure of the enzyme showing presence of a signal peptide (SP) indicating its extracellular nature, catalytic domain (CD), substrate binding domain (SBD) and linker domain (LD) b site for cleavage of the signal peptide between amino acids 43 and 44 as predicted by SignalP (marked with red arrow)

Fig. 2

Multiple sequence alignment of the PHB depolymerase amino acid sequence from N. dassonvillei NCIM 5124 (MCK9871921.1) with other actinomycetes [N. dassonvillei DSM 43111 (ADH65500.1), Nocardiopsis aegyptia (WP_246407222.1), Nocardiopsis alba (AFR07191.1), Micromonospora sp. (ADU09202.1), Janibacter sp. (UTT65431.1), Thermobifida fusca (EOR72938.1), Streptomyces ascomycinicus (AAF86381.1)] using Clustal W represented via ESPript 3

Fig. 3

Growth characteristics and degradation of PHB granules by N. dassonvillei NCIM 5124. Growth of the culture on a GYM agar plate showing characteristic morphology b MSM-PHB agar showing clearance zone indicative of PHB degradation. Visual observations of degradation in liquid MSM-PHB c uninoculated medium showing turbidity due to presence of intact PHB d medium inoculated with the culture showing clearance e top view of flask showing growth of the culture [white single arrows point to clearance and white double arrows point to growth of the organism]

Fig. 4

Comparative study on morphological features of N. dassonvillei NCIM 5124 on different carbon sources monitored by FE-SEM analysis. Microscopic observations of the culture a,b grown on PHB, c,d cultivated on glucose (magnification a,c: ×30,000; b,d: ×50,000)

Fig. 5

Effect of different parameters on PHB depolymerase activity in N. dassonvillei NCIM 5124 a enzyme activity over a period of time [inset: optical microscope images of C: control uninoculated samples with visible granules of PHB indicated by black single arrow; T: samples inoculated with the culture showing extensive development of mycelia marked by black double arrows] b effect of different concentrations of NaCl on the production of the enzyme (* indicates significant difference with P ≤ 0.05)

Fig. 6

Effect of different parameters on production of PHB depolymerase by N. dassonvillei NCIM 5124 in Bushnell Hass Medium using OVAT approach. Influence of a PHB content—0.1, 0.2, 0.3% b size of inoculum—2, 5, 8% c agitation—110, 120, 130 rpm d pH—6.0, 7.5, 9.0 e temperature—20, 30, 40 °C f ammonium nitrate—0.1, 0.15, 0.2% on enzyme activity. [Bars indicate average values with error bars representing standard deviation and asterisks (*) depict significant difference with P ≤ 0.05]

Fig. 7

Graphical representation of growth and degradation of PHB under optimized conditions. Growth (black circle) estimated in terms of cell dry weight (g/L) and PHB degradation (black triangle) monitored in terms of decrease in absorbance of medium at 600 nm (A₆₀₀) after settlement of biomass for 15 min

Fig. 8

FE-SEM observations indicating degradation of PHB, PHBV and PHBVH by N. dassonvillei. Representative images of a–c intact films of PHB as control samples; d–f PHB films after degradation; g–i undegraded films of PHBV; j–l PHBV films after degradation; m–o undamaged films of PHBVH; p–r PHBVH films after degradation (magnification a,d,g,j,m,p: ×250; b,e,h,k,n,q: ×1000; c,f,i,l,o,r: ×5000; white single arrows point towards pits and burrows in films due to degradation; white double arrows indicate mycelial growth)

Fig. 9

Degradation of PHB and copolymer films monitored in terms of weight loss. The graph depicts average weight (in mg) of PHB (red bars), PHBV (blue bars) and PHBVH (grey bars) over a period of time