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Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser155, Asp253, and His282 residues. The catalytic efficiency (kcat/Km) of Est19 for the pNPC6 substrate is 148.68 s−1mM−1 at 40 °C. Replacing Glu154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10–60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis , on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties. Human gut bacteria can degrade arabinoxylans, polysaccharides found in dietary fiber. Here, Pereira et al. identify a bacterial gene cluster encoding esterases for degradation of complex arabinoxylans. The action of these enzymes results in accumulation of ferulic acid, a phenolic compound with antioxidative and immunomodulatory properties.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling 1 , but reduced secretion of WNT ligands increases the ability of Apc -mutant intestinal stem cells to colonize a crypt (known as fixation) 2 . Here we investigated how Apc -mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc -mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc -mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc -mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc -mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer. NOTUM from Apc -mutant cells acts as a key mediator during the early stages of mutation fixation and drives the formation of intestinal adenomas.

EDITOR'S NOTE Readers are alerted that there is currently a discussion regarding the use of some of the unpublished genomic data presented in this manuscript. Appropriate editorial action will be taken once this matter is resolved. Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota , and Zygomycota . Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also revealed a complex history of lineage-specific expansions and attritions for the PL1 family. Conclusions Our study provides insights into the variety and expansion of fungal CAZyme classes and revealed the relationship of CAZyme size and diversity with their nutritional strategy and host specificity.

Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~10 7 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired ( S )-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity. Optimizing an enzyme usually requires testing thousands of variants, thus consuming large amounts of material and time. Here, the authors present a method that allows for measuring two different activities of the same enzyme simultaneously instead of doing two consecutive rounds of screening.

Background Yaks are able to utilize the gastrointestinal microbiota to digest plant materials. Although the cellulolytic bacteria in the yak rumen have been reported, there is still limited information on the diversity of the major microorganisms and putative carbohydrate-metabolizing enzymes for the degradation of complex lignocellulosic biomass in its gut ecosystem. Results Here, this study aimed to decode biomass-degrading genes and genomes in the yak fecal microbiota using deep metagenome sequencing. A comprehensive catalog comprising 4.5 million microbial genes from the yak feces were established based on metagenomic assemblies from 92 Gb sequencing data. We identified a full spectrum of genes encoding carbohydrate-active enzymes, three-quarters of which were assigned to highly diversified enzyme families involved in the breakdown of complex dietary carbohydrates, including 120 families of glycoside hydrolases, 25 families of polysaccharide lyases, and 15 families of carbohydrate esterases. Inference of taxonomic assignments to the carbohydrate-degrading genes revealed the major microbial contributors were Bacteroidaceae , Ruminococcaceae , Rikenellaceae , Clostridiaceae , and Prevotellaceae . Furthermore, 68 prokaryotic genomes were reconstructed and the genes encoding glycoside hydrolases involved in plant-derived polysaccharide degradation were identified in these uncultured genomes, many of which were novel species with lignocellulolytic capability. Conclusions Our findings shed light on a great diversity of carbohydrate-degrading enzymes in the yak gut microbial community and uncultured species, which provides a useful genetic resource for future studies on the discovery of novel enzymes for industrial applications.

Bacillus subtilis as an important host has been widely used in synthetic biology, metabolic engineering, and production of industrial enzymes. To fully take advantage of this organism, 114 endogenous putative promoters were measured with a green fluorescent protein reporter and four classes of phase-dependent promoters (class I: exponential phase; class II: middle-log and early stationary phases; class III: lag-log and stationary phases; class IV: stationary phase) with different strengths were identified. The transcriptional strengths ranged from 0.03 to 2.03-fold of that of the commonly used strong promoter P 43 . On this basis, the temperature (for instance P bltD , P ydaD , and P gerBC ) and pH (such as P abrB , P ydjO , and P opuE ) inducible phase-dependent promoters were further identified and characterized. In comparison, P abrB (class I), P spoVG (class II), and P lytR (class III) achieved the highest expression levels of esterase, keratinase, and alkaline polygalacturonate lyase, respectively. The constructed phase-dependent promoter library should have great application potentials for enzyme production, metabolic engineering, and synthetic biology.

Background GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP ( Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. Results In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes. Conclusions Our current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study.