Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.
The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an ␣-amylase (amyA), a 1,4-␣-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.
The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to -lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (؉)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.Modern biotechnology has a steadily increasing demand for novel biocatalysts, thereby prompting the development of novel experimental approaches to find and identify novel biocatalyst-encoding genes. Recently, there has been an increase in the number of studies using a metagenomic approach to investigate the catalytic potential of uncultured microorganisms (8, 28). The term metagenome was introduced to describe the genomes of complex microbial communities found in natural habitats, only a small fraction of which can be cultured (1, 17). Investigation of metagenomes became possible after the development of strategies for the isolation and cloning of environmental DNA (41, 47). Modern metagenomic developments and cloning strategies have recently been reviewed in detail (8,9,16,46). Once constructed, metagenomic libraries can be screened for a wide range of ecologically and biotechnologically interesting phenotypes (44).In the search for novel biocatalysts, there are various metagenomic strategies that are used for targeting specific catalyst characteristics such as substrate range or temperature and pH optima. One approach is to generate the metagenomic library from soils or sediments that are known to harbor a high level of microbial diversity and thus, potentially, a wide diversity of biocatalysts (7). This approach has been used successfully to find a wide variety of novel catalysts and secondary metabolites (9,19,20,27,30,39). A further development of this approach is to create the metagenomic library from an environment that has been subjected to extreme conditions in the likelihood that enzymes from such an environment will be able to f...
The oxidative lactonization of 1,4-, 1,5-, and 1,6-diols using horse liver alcohol dehydrogenase (HLADH) is reported. Molecular oxygen was used as terminal electron acceptor by utilization of the laccase-mediator concept to regenerate the oxidized nicotinamide cofactor and producing water as sole byproduct. Spontaneous hydrolysis of the lactone products was identified as a major limiting factor toward preparative application of the system, which can be alleviated by using a two liquid phase approach to extracting the product into an organic solvent.
Highly productive biocatalytic reductions were established using an isolated alcohol dehydrogenase (ADH) under water‐deficient conditions. First, a solvent‐free system was evaluated for the reduction of 2‐butanone catalyzed by ADH evo‐1.1.200 promoted by the “smart cosubstrate” 1,4‐butanediol. ADH evo‐1.1.200 excelled by its activity and stability under high reagent concentrations and hence was the enzyme of choice. However, conversion of 2‐butanone was limited to <1 % in 10 days under the solvent‐free conditions. Therefore, water‐immiscible organic solvents were evaluated whereby the highest conversions were achieved in MTBE and toluene. MTBE was chosen as its different boiling point compared to other reaction components (e.g., 2‐butanone, 2‐butanol, diol cosubstrate, and lactone coproduct) would simplify the downstream processing. Further on, by tuning substrate loading, the productivity of the ADH evo‐1.1.200 was successfully increased to a turnover number (TON) of 64 000.
Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity. In this study, we identified novel PET binding CBMs and characterized the CBM-PET interplay. We developed a semi-quantitative method to detect CBMs bound to PET films. Screening of eight CBMs from diverse families for PET binding revealed one CBM that possesses a high affinity towards PET. Molecular dynamics (MD) simulations of the CBM–PET interface revealed tryptophan residues forming an aromatic triad on the peptide surface. Their interaction with phenyl rings of PET is stabilized by additional hydrogen bonds formed between amino acids close to the aromatic triad. Furthermore, the ratio of hydrophobic to polar contacts at the interface was identified as an important feature determining the strength of PET binding of CBMs. The interaction of CBM tryptophan residues with PET was confirmed experimentally by tryptophan quenching measurements after addition of PET nanoparticles to CBM. Our findings are useful for engineering PET hydrolyzing enzymes and may also find applications in functionalization of PET. Electronic supplementary material The online version of this article (10.1007/s00253-019-09760-9) contains supplementary material, which is available to authorized users.
is shown to be an efficient cosubstrate to promote NAD(P)H-dependent redox biocatalysis. The thermodynamically and kinetically inert lactone coproduct makes the regeneration reaction irreversible. Thereby not only the molar surplus of cosubstrate is dramatically reduced but also faster reaction rates are obtained. Scheme 1 Comparison of the 'classical' biocatalytic MPV-reduction e.g. using isopropanol and the proposed 'smart cosubstrate' approach using 1,4-butanediol. The lactone coproduct renders the regeneration reaction irreversible. The lower part shows the equilibrium conversions as calculated from the law of mass action (lines) and the waste generated (bars). † Electronic supplementary information (ESI) available: Detailed description of the experimental and analytical procedures and further experimental data. See
Gamma-aminobutyric acid (GABA), a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods, and the biodegradable plastic polyamide 4. Corynebacterium glutamicum shows great potential for the production of GABA from glucose. GABA added to the growth medium hardly affected growth of C. glutamicum, since a half-inhibitory concentration of 1.1 M GABA was determined. As alternative to GABA production by glutamate decarboxylation, a new route for the production of GABA via putrescine was established in C. glutamicum. A putrescine-producing recombinant C. glutamicum strain was converted into a GABA producing strain by heterologous expression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) genes from Escherichia coli. The resultant strain produced 5.3 ± 0.1 g L of GABA. GABA production was improved further by adjusting the concentration of nitrogen in the culture medium, by avoiding the formation of the by-product N-acetylputrescine and by deletion of the genes for GABA catabolism and GABA re-uptake. GABA accumulation by this strain was increased by 51 % to 8.0 ± 0.3 g L, and the volumetric productivity was increased to 0.31 g L h; the highest volumetric productivity reported so far for fermentative production of GABA from glucose in shake flasks was achieved.
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