β-Mannanases are involved in the hydrolysis of mannan, a major component of hemicellulose. The genome of Glutamicibacter halophytocola sp., an endophytic bacterium isolated from Crithmum maritimum, a halophilic plant from the Island of Crete, encodes a putatively secreted β-mannanase (Gh_GH26) belonging to glycoside hydrolase 26 family (GH26). This sequence occupies a poorly explored region of the GH26 sequence space. Gh_GH26 consists of three domains, including a C-terminal carbohydrate-binding module 23 (CBM23), which is uncommon among GH26s and has a peculiar amino acidic composition at the -3/-4 substrate subsites. Gh_GH26 displays halophilic behavior, retaining activity up to 2.5 M NaCl, and exhibits endo-mannanase activity. Functional analyses combined with structural predictions suggest that Gh_GH26 coordinates Ca2+ ions through a single binding site located within the CBM23 domain. The residues involved in Ca2+ binding are conserved in the CBMs of other characterized GH26s, despite their low similarity with Gh_GH26. Site-directed mutagenesis shows that Ca2+ ions contribute to both enzyme activity and stability. These results suggest that Ca2+ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain. Overall, this work expands our understanding of the functional and structural diversity of the GH26 family, highlighting its potential for application in the degradation of mannan-containing biomass under saline conditions.
M. Orlando, Mattia Salvadori, P. Sarris et al.· International Journal of Bio...· 0 citations
BACKGROUND
Polyethylene (PE) is the most widely produced polyolefin and represents a major contributor to plastic waste, largely due to its chemical stability and resistance to biological degradation. PE biodegradation is typically characterized as a series of oxidative metabolic processes rather than mineralization. An urgent priority is the development of integrated experimental setups that bring together physiological, biochemical, and molecular analyses under controlled cultivation conditions to unlock the metabolic traits of PE biodegradation. This study aims to elucidate the molecular mechanisms of commercial low-molecular-weight PE (LDPE) attack by Rhodococcus opacus R7 through an integrated genome-to-function approach, including growth and extracellular laccase assays, intracellular lipid quantification, gas chromatography coupled with mass spectrometry (GC-MS) analysis of compounds associated with untreated LDPE, and transcriptional profiling of oxidative enzymes.
RESULTS
LDPE utilization was evaluated under multiple cultivation strategies (1% single-dose vs. 0.4% fed-batch), inoculum conditions, and polymer types (untreated or UV-pretreated) over 28 days. Fed-batch LDPE (0.4%) supported slightly higher viable cell numbers than fixed-dose LDPE (1%), while prolonged PE pre-adaptation did not enhance growth. Extracellular laccase activity was detected under all conditions, but was more influenced by the physiological status of the inoculum than by PE concentration or pretreatment. Lipid accumulation was early detected in case of 1% PE, while the fed-batch conditions reflected the growth phase and physiological responses. GC-MS analyses revealed that LDPE oxidation products vary compared to abiotic and control samples. Specifically, fixed-dose LDPE favored a progressive change in the pattern-profile of carboxylic acids and medium- to long-chain alkanes, while fed-batch LDPE produced a heterogeneous mixture, including alcohols and ketones, consistent with a continuous and asynchronous transformation of the polymer. The fed-batch condition induced a broader and earlier transcriptional activation of oxidative genes, including seven laccase-like multicopper oxidases (LMCOs), alkane monooxygenase (alkB), benzoate dioxygenase (benA), and cytochrome P450 hydroxylase. Long-chain n-alkanes, particularly tetracosane (C24), strongly induced oxidative enzymes, suggesting their role as metabolic signals during PE degradation.
CONCLUSIONS
Overall, LDPE attack by R. opacus R7 emerges as a dynamic process shaped by substrate accessibility and cultivation strategy, highlighting its potential as a platform for controlled plastic biodegradation.
J. Zampolli, Mattia Salvadori, Valentina Vincenti et al.· BMC Microbiology· 0 citations
