Marine algae are responsible for half of the global primary production, converting carbon dioxide into organic compounds like carbohydrates. Particularly in eutrophic waters, they can grow into ...massive algal blooms. This polysaccharide rich biomass represents a cheap and abundant renewable carbon source. In nature, the diverse group of polysaccharides is decomposed by highly specialized microbial catabolic systems. We elucidated the complete degradation pathway of the green algae-specific polysaccharide ulvan in previous studies using a toolbox of enzymes discovered in the marine flavobacterium Formosa agariphila and recombinantly expressed in Escherichia coli. In this study we show that ulvan from algal biomass can be used as feedstock for a biotechnological production strain using recombinantly expressed carbohydrate-active enzymes. We demonstrate that Bacillus licheniformis is able to grow on ulvan-derived xylose-containing oligosaccharides. Comparative growth experiments with different ulvan hydrolysates and physiological proteogenomic analyses indicated that analogues of the F. agariphila ulvan lyase and an unsaturated beta-glucuronylhydrolase are missing in B. licheniformis. We reveal that the heterologous expression of these two marine enzymes in B. licheniformis enables an efficient conversion of the algal polysaccharide ulvan as carbon and energy source. Our data demonstrate the physiological capability of the industrially relevant bacterium B. licheniformis to grow on ulvan. We present a metabolic engineering strategy to enable ulvan-based biorefinery processes using this bacterial cell factory. With this study, we provide a stepping stone for the development of future bioprocesses with Bacillus using the abundant marine renewable carbon source ulvan.
Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases ...due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) — a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1:
Bilophilia wadsworthia,
TA-5:
Halomonas elongata
, TA-9:
Burkholderia cepacia
, and TA-10:
Burkholderia multivorans
) were obtained in a soluble form as tetramers in
E. coli
. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from
Burkholderia multivorans
features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C.
Key points
•
Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily
.
•
The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C
.
•
A tetrameric structure is not necessarily associated with a higher operational stability
Graphical abstract
Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more ...environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.
Enzymatic hydrolysis holds great promise for plastic waste recycling and upcycling. The interfacial catalysis mode, and the variability of polymer specimen properties under different degradation ...conditions, add to the complexity and difficulty of understanding polymer cleavage and engineering better biocatalysts. We present a systemic approach to studying the enzyme-catalyzed surface erosion of poly(ethylene terephthalate) (PET) while monitoring/controlling operating conditions in real time with simultaneous detection of mass loss and changes in viscoelastic behavior. PET nanofilms placed on water showed a porous morphology and a thickness-dependent glass transition temperature (Tg) between 40°C and 44°C, which is >20°C lower than the Tg of bulk amorphous PET. Hydrolysis by a dual-enzyme system containing thermostabilized variants of Ideonella sakaiensis PETase and MHETase resulted in a maximum depolymerization of 70% in 1 h at 50°C. We demonstrate that increased accessible surface area, amorphization, and Tg reduction speed up PET degradation while simultaneously lowering the threshold for degradation-induced crystallization.
Display omitted
•The Tg of PET in contact with water decreases to 40°C in nanometric films•PET turnover rate decreases above 50°C with a thermostable IsPETase variant•Incomplete PET degradation coincides with surface deposition of deactivated enzymes•Degradation-induced surface crystallization is not seen in thin films up to 60°C
While the majority of plastic waste is landfilled, incinerated, or recycled—in that order—a large portion of ∼29% has an unknown fate: about 4% reaches the oceans, and other large amounts enter the biosphere as micro- and nanoplastics derived from environmental degradation. In a short period of time, microorganisms may have evolved various mechanisms to address these xenobiotics, such as biocatalytic depolymerization of plastics and assimilation of their monomers. Our research reveals that surface-layer degradation plays a crucial role in biocatalytic PET depolymerization. The change in physicochemical properties of a nanoscale thin film during biocatalytic degradation was monitored to mimic the surface-erosion process of bulk polymers. Our findings will strengthen the mechanistic understanding of biocatalytic polymer degradation in general and may serve as the basis for optimizing bioremediation strategies (e.g., for accumulated plastic pollution in wastewater treatment plants).
Enzymes can mostly access the surface layer of PET during depolymerization. We show that an amorphous PET thin film with comparable properties to the PET surface layer has a lower glass transition temperature (Tg) of 40°C than bulk PET’s Tg of 65°C–81°C. As a result, within a few hours at 40°C–50°, this PET thin film was rapidly depolymerized by an IsPETase variant. Our findings will be beneficial for industrial implementation of enzymatic plastic degradation using properly treated polymers.
Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6‐O‐methyl‐d‐galactose, an abundant monosaccharide in the red algal ...polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.
The oxidative demethylation of 6‐O‐methyl‐d‐galactose produces formaldehyde. Marine bacteria that utilize this sugar in the degradation of porphyran must therefore possess suitable formaldehyde detoxification pathways. The connection between the degradation of marine carbohydrates and the detoxification of formaldehyde has been demonstrated for the marine model organism Zobellia galactanivorans.
Marine algae produce complex polysaccharides, which can be degraded by marine heterotrophic bacteria utilizing carbohydrate-active enzymes. The red algal polysaccharide porphyran contains the methoxy ...sugar 6-
O
-methyl-
d
-galactose (G6Me). In the degradation of porphyran, oxidative demethylation of this monosaccharide towards
d
-galactose and formaldehyde occurs, which is catalyzed by a cytochrome P450 monooxygenase and its redox partners. In direct proximity to the genes encoding for the key enzymes of this oxidative demethylation, genes encoding for zinc-dependent alcohol dehydrogenases (ADHs) were identified, which seem to be conserved in porphyran utilizing marine
Flavobacteriia
. Considering the fact that dehydrogenases could play an auxiliary role in carbohydrate degradation, we aimed to elucidate the physiological role of these marine ADHs. Although our results reveal that the ADHs are not involved in formaldehyde detoxification, a knockout of the ADH gene causes a dramatic growth defect of
Zobellia galactanivorans
with G6Me as a substrate. This indicates that the ADH is required for G6Me utilization. Complete biochemical characterizations of the ADHs from
Formosa agariphila
KMM 3901
T
(FoADH) and
Z. galactanivorans
Dsij
T
(ZoADH) were performed, and the substrate screening revealed that these enzymes preferentially convert aromatic aldehydes. Additionally, we elucidated the crystal structures of FoADH and ZoADH in complex with NAD
+
and showed that the strict substrate specificity of these new auxiliary enzymes is based on a narrow active site.
Key points
• Knockout of the ADH-encoding gene revealed its role in 6-O-methyl-D-galactose utilization, suggesting a new auxiliary activity in marine carbohydrate degradation.
• Complete enzyme characterization indicated no function in a subsequent reaction of the oxidative demethylation, such as formaldehyde detoxification.
• These marine ADHs preferentially convert aromatic compounds, and their strict substrate specificity is based on a narrow active site.
Polyethylene terephthalate (PET) is a mass-produced petroleum-based synthetic polymer. Enzymatic PET degradation using, for example,
PETase (
PETase) can be a more environmentally friendly and ...energy-saving alternative to the chemical recycling of PET. However,
PETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (
) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error-prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of
PETase. The library was screened against the commercially available polyester-polyurethane Impranil DLN W 50 for more thermostable
PETase variants, yielding four variants with higher melting points. The most promising
PETaseTM
variant had a 5.0°C higher melting point than
PETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published
PETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.
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