Extracellular Esterase Activity Research Articles

Multiple strategies to improve extracellular secretion and activity of feruloyl esterase

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5′ UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities during Impranil biodegradation by Alicycliphilus denitrificans BQ1.

The concerted action of commercial esterases, proteases and amidases has been demonstrated to be relevant in polyurethane (PU) degradation by in vitro experiments. However, the spatial and temporal dynamics of these activities during PU biodegradation by PU-degrading bacteria have not been addressed. Here, we examined the capability of Alicycliphilus denitrificans BQ1 to biodegrade the polyester (PS)-PU Impranil, analyzed the temporal and spatial coordination between the extracellular and cytoplasmic esterase and urethane-cleaving activities, and their independent and combined effects on Impranil biodegradation. A. denitrificans BQ1 grew in Impranil, and its clearing was correlated with the cleavage of ester and urethane groups since early times, with decrements of some Impranil compounds and the appearance of biodegradation products. While extracellular esterase was active at early times with its maximum at 18h, urethanase appeared at this time and increased up to the end of the analysis (48h), with the cytoplasmic activities behaving similarly but with lower levels than the extracellular ones. Both enzymatic activities exhibited distinct substrate specificity depending on their cellular localization and cultivation times, suggesting they cleave differentially located groups. As the urethane cleavage occurred since early times, when no urethane-cleaving activity was detected, different proteins should be acting at early and late times. In vitro experiments with independent or combined cellular protein fractions supported the previous deduction and confirmed the concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities. A two-stage process for Impranil degradation by A. denitrificans BQ1 is proposed.

Exploring the polyurethanolytic activity and microbial composition of landfill microbial communities.

The microbial composition of polyurethane degrading communities has been barely addressed, and it is unknown if microenvironmental conditions modify its composition, affecting its biodegradative capacity. The polyurethanolytic activity and taxonomic composition of five microbial communities, selected by enrichment in the polyether-polyurethane-acrylic (PE-PU-A) coating PolyLack®, from deteriorated PU foams collected at different microenvironments in a municipal landfill (El Bordo Poniente, BP) were explored. All BP communities grew similarly in PolyLack® as the sole carbon source, although BP1, BP4, and BP5 showed better performance than BP2 and BP7. FTIR spectroscopy showed that ester, urethane, ether, aromatic and aliphatic groups, and the acrylate component were targets of the biodegradative activity. Extracellular esterase activity was higher at 5days of cultivation and decreased at 21days, while urease activity showed the opposite. Microbial composition analysis, assessed by 16S rDNA V3 region PCR-DGGE, revealed a preponderance of Rhizobiales and Micrococcales. The reported PU-degrading genera Paracoccus, Acinetobacter, and Pseudomonas were identified. In contrast, Advenella, Bordetella, Microbacterium, Castellaniella, and Populibacterium, some of them xenobiotics degraders, can be considered potentially PU-degrading genera. Correspondence analysis identified independent groups for all communities, except the BP4 and BP5. Although partial taxonomic redundancy was detected, unique OTUs were identified, e.g., three members of the Weeksellaceae family were present only in the BP4/BP5 group. These results suggest that the microenvironmental conditions where the landfill microbial communities were collected shaped their taxonomical composition, impacting their PE-PU biodegradative capacities. These BP communities represent valuable biological material for the treatment of PU waste and other xenobiotics. KEY POINTS: • Landfill microbial communities display slightly different capacities for growing in polyether-polyurethane-acrylic. • Ester, urethane, ether, aromatic, aliphatic, and acrylate groups were attacked. • Esterase activity was more significant at early culture times while urease activity at latter. • Landfill microenvironments shape partial taxonomical redundancy in the communities. • Best communities' performance seems to be related to unique members' composition.

SELECTION OF AUTOCHThONOUS sACCHAROMYCES AND NON-sACCHAROMYCES YEASTS STRAINS ACCORDING TO THEIR EXTRACELLULAR ENZIMATIC ACTIVITY

The aim of the present study was to investigate the production of extracellular enzymes in a number of twenty six autochtonous Saccharomyces and nonSaccharomyces strains selected in Dealu Mare region for wine production. The strains were screened for the production of extracellular b-glucosidase, esterase, pectinase and protease activity by inoculation the yeast strains onto selective media. All Saccharomyces tested strains showed at least two enzymatic activities while nonSaccharomyces strains showed activity at least for one enzyme. The weakest activity was recorded in case of β-glucosidase. Most of the tested strains exhibit more or less intense activity for polygalacturonase/pectinase and protease. This study put into evidence the potential of autochtonous and especially of non-Saccharomyces strains as source of production of secondary compounds which can play an important role in improving the quality of wines.

Hydrolytic enzymes as probable virulence factors for Aspergillus ochraceus fm90 in aspergillosis

THE MAIN concept of this study was to assess the potentiality of hydrolytic enzymes as virulence factors for Aspergillus species during aspergillosis infection process. Forty Aspergillus species were isolated from medical (15 isolates) as well as environmental isolates (25 isolates) from soils, outdoor and indoor environments. Extracellular proteases, phospholipases and esterase activities were measured. The pathogenicity of some Aspergillus species was in vivo assessed. It was represented in mean survival times, mortality percentages, fungal counts in different organs, and histopathological examination of lung tissue. The expression levels of protease, phospholipase and esterase genes were studied by real time PCR. Four physiologically significant isolates were selected out of forty and were identified up to molecular level. Aspergillus ochraceus fm90 recorded the highest pathogenicity as represented by mortality percentages and mean survival times of mice, while A. flavus fm90 was the least pathogenic one. Expression of genes of protease, phospholipase and esterase was found to be greater in A. ochraceus fm90 than in A. flavus fm90. It can be concluded that pathogenicity is probably related to physiological (enzymatic) activities of the isolates. Also, variation in expression levels of protease and phospholipase genes in A. ochraceus fm90 and A. flavus fm90 could denote their possible involvement in the pathogenicity process.

Biodegradation of polyacrylic and polyester polyurethane coatings by enriched microbial communities.

Microbial communities are more effective in degrading natural polymers and xenobiotics than pure cultures. Biodegradation of polyacrylic and polyurethane polymers by bacterial and fungal strains has been addressed, but limited information about their biodegradation by microbial communities exists. The aim of this work was to evaluate the ability of three enriched microbial communities (BP1h, BP3h, and BP7h), selected from deteriorated foam pieces collected in a landfill, to biodegrade the polyacrylic component of the 2K-PU coating Bayhydrol® A2470 and the polyester polyurethane coating NeoRez™ R-9637. Two communities were further selected to quantify extracellular esterase, protease, and urease activities, to identify their taxonomic composition, and to analyze the ability of their isolated members to grow in those polymers. The growth of the three communities was larger in polyester polyurethane than in polyacrylic and their biodegradative activities affected ester, urethane, ether, aromatic, and aliphatic groups of the compounds present in the coatings. From all the communities growing in polyacrylic or in polyester polyurethane, two and five different types of colonies were isolated, respectively. In polyacrylic, extracellular esterase and protease activities were at their maximum level at 7days of culture, whereas in polyester polyurethane, protease and urease were greatest at 21days. All the isolated community members were identified as xenobiotics degraders. The complete communities grew better in media with the polymers than the isolated members. This is one of the few studies reporting biodegradation of synthetic polymers by microbial communities and serves as basis for developing synthetic consortia with enhanced degradative abilities.

Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion

Context: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases.Objective: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein.Materials and methods: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity.Results: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity.Discussion: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases.Conclusion: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.

A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activity

Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (Rs pCAE: 3245UL−1) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs pCAE resulted in the efficient release of p-coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass.

Purification, amino acid sequencing and thermostability of an extracellular low molecular weight esterase produced by Bacillus subtilis NRRL 41270 in fermentation

Extracellular esterase activity in Bacillus subtilis NRRL 41270 fermentation broths was found to reside in a small protein with a molecular weight less than 10 kDa. Following purification, esterase activity on fluorescein dibutyrate was estimated at 12 U/min/mg proteins. Enzyme saturation was observed at 5 μM substrate concentration. The produced esterase hydrolysed tributyrin. Its specific activity was estimated to be 17.8 μmol acid released/min/mg proteins. The small protein was subjected to size exclusion chromatography, SDS-PAGE and amino acid sequencing. Analysis revealed a sequence of the following amino acid residues: eevaetysfyhitphdystshispapvqffspap, according to which the molecule has 34 amino acid residues and a calculated molecular mass of 3853, which was in accordance with the gel filtration and SDS-PAGE results. Sequence based analysis and use of bioinformatics tools showed no significant similarity with known proteins while revealed a strongly hydrophobic molecule, with a α-helical conformation in the N-terminal, the rest of the molecule being β-sheet-rich. The enzyme appeared to be thermostable with more than 85% of the original activity maintained after 120 h incubation at 60°C. The producer organism and the features of the micro enzyme, suggest the case of a biotechnologically interesting biocatalyst that should be further researched in terms of its stability and production characteristics.

Degradation of polyester polyurethane by an indigenously developed consortium of Pseudomonas and Bacillus species isolated from soil

This work investigated the degradation efficiency of bacterial co-culture against polyester polyurethane in comparison to the individual strains. For this purpose, a consortium of two already reported polyester polyurethane (PU) degrading bacterial strains Bacillus subtilis MZA-75 and Pseudomonas aeruginosa MZA-85 was developed. The degradation was analyzed through measurement of weight loss, scanning electron microscopy (SEM), fourier transformed infra-red (FT-IR) spectroscopy and Sturm test (CO2 evolution test). Polyester PU degradation was augmented in the presence of co-culture, as indicated by maximum weight reduction in Polyester PU film pieces within 20 days. SEM micrographs showed surface changes in the polyester PU films in the form of pits or holes that resides bacterial cells. Besides degradation of soft segment, a new peak at 2250 cm−1 appeared in FTIR spectrum, which corresponds to free isocyanates due to breakdown of urethane linkages. The quantity of carbon dioxide evolved in Sturm test was much higher due to high degradation rate in co-culture vessel as compared to control as well as the separate cultures. A sharp increase in extracellular esterase activity of co-culture against polyester PU was observed. Butanediol (BD) and Adipic Acid (AA) were detected as degradation products in relatively higher intensity than those observed in pure culture, through gas chromatography-mass spectrometry (GC-MS). Hence, it is concluded that consortium might be better than pure cultures in polyester PU waste management and used as a tool for recycling purified monomers (biochemical depolymerisation/monomerization).

A novel extracellular cold-active esterase of Pseudomonas sp. TB11 from glacier No.1: Differential induction, purification and characterisation

The production, purification, characterization and application of a novel cold active esterase by Pseudomonas sp. TB11 are described herein. A new finding regarding the production of extracellular esterase activity depending upon single cream as an inducer used for growth was investigated in this study. The crude esterase was subjected to a three-step enzyme purification, which resulted in a 15.21-fold purification and the specific activity of the final purified esterase increased to 1526.2U/mg protein and purified EstTB11 had a molecular mass of 65kDa. The N-terminal sequence of ten amino acids were: GVYDYKNLTT. Peptide mass finger printing revealed that some peptides showed homologues sequences (29%) to polyurethanase of Pseudomonas sp. FH4. Furthermore, the enzyme displayed the optimum pH of 8.5 and optimum temperature of 25°C and significantly high stability at 15–35°C for 72h. The enzyme was incubated with different metal ions at concentrations of 5 and 10mM, the activity of esterase was increased in the presence of K+, Na+ and Mg2+ and decreased with Ca2+, Al3+, Mn2+, and Fe3+. Experiments indicated that EstTB11 could hydrolyze milk fat to produce short and medium-chain fatty acid and this result layed the foundation for the application in increased aroma of milk products.

Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk

Heat-resistant enzymes of psychrotolerant microorganisms are associated with the spoilage of UHT-milk and milk products. In this study, we investigated the extracellular peptidase, esterase and lipase activity after submerged cultivation at 6 °C of 231 recently isolated microorganisms in milk medium. In contrast to the widely used agar diffusion tests for secretion of hydrolases, here a more realistic liquid screening approach was used. The advantages of the latter are the possibility of quantifying the enzyme activity in volumetric units and the opportunity to simulate the growth conditions of the microorganisms to the storage conditions of raw milk. The majority of enzymatically active isolates belonged to Gram-negative bacteria, especially the genus Pseudomonas. Surprisingly, among them, twelve novel Pseudomonas species were discovered. In this study, we demonstrated that numerous raw milk isolates, including bacteria and yeasts, produce extracellular enzymes that may cause spoilage problems for the dairy industry.

Quorum-Sensing Mechanisms Mediated by Farnesol in Ophiostoma piceae: Effect on Secretion of Sterol Esterase.

Ophiostoma piceae CECT 20416 is a dimorphic wood-staining fungus able to produce an extracellular sterol-esterase/lipase (OPE) that is of great biotechnological interest. In this work, we have studied the morphological change of this fungus from yeast to hyphae, which is associated with the cell density-related mechanism known as quorum sensing (QS), and how this affects the secretion of OPE. The data presented here confirm that the molecule E,E-farnesol accumulates as the cell number is growing within the population. The exogenous addition of this molecule or spent medium to the cultures increased the extracellular activity of OPE 2.5 times. This fact was related not to an increase in microbial biomass or in the expression of the gene coding for OPE but to a marked morphological transition in the cultures. Moreover, the morphological transition also occurred when a high cell density was inoculated into the medium. The results suggest that E,E-farnesol regulates through QS mechanisms the morphological transition in the dimorphic fungus O. piceae and that it is associated with a higher extracellular esterase activity. Furthermore, identification and transcriptional analysis of genes tup1 and cyr1, which are involved in the response, was carried out. Here we report enhanced production of a sterol-esterase/lipase of biotechnological interest by means of QS mechanisms. These results may be useful in increasing the production of secreted enzymes of other dimorphic fungi of biotechnological interest.

Degradation of Polyurethane by Aspergillus flavus (ITCC 6051) Isolated from Soil

The present study deals with the isolation of fungi from soil with the ability to degrade polyurethane (PU). A pure fungal isolate was analyzed for its ability to utilize PU as a sole carbon source in shaking culture for 30 days. Incubation of PU with Aspergillus flavus resulted in 60.6% reduction in weight of PU. The scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR) results showed certain changes on the surface of PU film and formation of some new intermediate products after polymer breakdown. Thermogravimetric curves showed changes between the thermal behavior of the samples that were inoculated with A. flavus and control. FTIR spectra showed detectable changes in control and incubated samples, suggesting that degradation occurs, with the decreased intensity of band at 1,715 cm(-1), corresponding to ester linkages. We have identified an extracellular esterase activity which might be responsible for the polyurethanolytic activity.

In Vitro Evaluation of Phospholipase, Proteinase, and Esterase Activities of Candida parapsilosis and Candida metapsilosis

The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR-RFLP) of the secondary alcohol dehydrogenase (SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue.

Degradation of poly (lactic acid) and nanocomposites by Bacillus licheniformis

The disposal problem due to non-degradable petroleum-based plastics has raised the demand for biodegradable polymers. The degradation of poly (lactic acid) (PLA) has been studied for several years, but the understanding of involved mechanisms is still incomplete. Based on our previous studies, and it is hypothesized an enzymatic involvement, the aim of this study was to continue investigations on the degradation of PLA and its nanocomposites by Bacillus licheniformis. Biodegradation of PLA and its nanocomposites (CLOISITE 30B and SOMASIF MEE) was performed on compression-molded, 25 × 25 × 0.6-mm films. Firstly, two plastic films were dipped into sterile nutrient broth inoculated with B. licheniformis and incubated at 32°C. Then, to verify if biodegradation was due to extracellular esterase, the culture broth was filtered to remove B. licheniformis cells, and the plastic materials were put into this broth. PLA degradation by B. licheniformis was accelerated by the presence of organoclays. After 5months in liquid culture, nanocomposites showed only the 10% of residual mass, compared with the 60% of pure PLA. Extracellular esterase activity was detected in the filtered culture broth confirming that PLA biodegradation was probably due to this enzyme action.

In vitro response of monocyte‐derived macrophages to a decellularized pericardial biomaterial

Decellularized tissue-derived heart valves are an example of biomaterials derived from natural scaffolds. These types of implants are increasing in popularity although their in vivo performance is still only poorly understood and has, at times, been catastrophic. It is apparent that better understanding is required before these biomaterials can be used safely. In this study, the human monocyte-derived macrophage (MDM) response to decellularized bovine pericardium (DBP) was used as a model to predict the biological performance of these materials on implantation. Human monocytes differentiated on tissue culture polystyrene (TCPS) for 14 days were trypsinized and reseeded onto DBP, TCPS, and polydimethylsiloxane (PDMS) for 48 h. The MDMs on DBP contained less intracellular and extracellular esterase activity compared with MDMs on TCPS and PDMS, as well as less acid phosphatase activity than on TCPS. As well, morphologically, MDMs on DBP were less spread, less multinucleated and did not display many lamellipodia. Taken together, these data represent the first evidence of the MDM response to intact, native extracellular matrix, demonstrating that these cells reacted with an altered, possibly reduced foreign body response on this natural scaffold compared with the two control surfaces. This in vitro MDM cell model may provide a novel method for predicting and elucidating the biological performance of tissue-derived biomaterials, thereby directing a more rational design of biomaterials for tissue regeneration purposes.

Media Formulation using Complex Organic Nutrients for Improved Activity, Productivity, and Yield of Candida rugosa Lipase and Esterase Enzymes

Candida rugosa is an excellent source of multiple lipase and esterase enzymes; therefore, it is of technological importance to formulate the medium that provides high activity for each enzyme. In this work, the cultivation medium comprising complex nutrients that provided the highest activity, productivity, and yield of C. rugosa enzymes individually was formulated. Time courses of the extracellular and intracellular lipase and esterase activities of C. rugosa were represented and the role of protease in the cultivation progress was discussed. Urea, soy-peptone, yeast extract, a mixture of soy-peptone and yeast extract, cheese whey, and wheat mill bran were tested for their lipolytic and esterasic activities. Urea provided considerably higher extracellular lipase activity when compared to other nitrogen sources; however, soy-peptone provided the highest extracellular esterase activity. Hazelnut, olive, sesame, soybean, and flax seed oils affected the enzyme activities to different extents related to their fatty acid compositions. Hazelnut oil and olive oil provided the highest extracellular lipase and esterase activities, respectively, whereas sesame oil produced the highest biomass. High C18 and C16 ester contents of vegetable oils promoted high lipase and esterase productions, respectively. A temperature of 30°C yielded the highest extracellular and intracellular lipase and esterase activities; however, 35°C produced the highest biomass.

Two glucuronoyl esterases of Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.