Arsenic, lead and cadmium concentration in food and estimated daily intake in the Cuban population and the health risks using a Total Diet Study.

This study estimates the intake of arsenic, lead and cadmium by the adult population (aged 18-91) of Cuba. The food consumption indices were obtained through 24-h dietary recall surveys applied to 450 people between October 2020 and March 2021. The Estimated Dietary Intake (EDI) of t-As (54.6 µg/day), Pb (118.5 µg/day) and Cd (35.1 µg/day) complied with Cuban legislation but was higher than the EDI for Cd established by the CONTAM Panel. The Target Hazard Quotients for the three contaminants were: iAs (0.220), Pb (0.409) and Cd (0.424), making the value of the Total Target Hazard Quotient 1.05, which indicates potential health risks for the population. Additionally, associated carcinogenic risks were: iAs (1.0·10-4), Pb (7.2·10-4) and Cd (25.9·10-4). Therefore, 10, 72 and 259 persons per 100,000 inhabitants are likely prone to developing cancer due to the ingestion of iAs, Pb and Cd, respectively.

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.

The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.

Penicillium purpurogenum produces a novel, acidic, GH3 beta-xylosidase: Heterologous expression and characterization of the enzyme.

Xylan, a component of plant cell walls, is composed of a backbone of ß-1,4-linked xylopyranosyl units with a number of substituents. The complete degradation of xylan requires the action of several enzymes, among them ß-xylosidase. The fungus Penicillium purpurogenum secretes a number of enzymes participating in the degradation of xylan. In this study, a ß-xylosidase from this fungus was expressed in Pichia pastoris, and characterized. This enzyme (Xyl2) is a member of glycoside hydrolase family 3; it consists of a sequence of 792 residues including a signal peptide of 20 residues, with a theoretical molecular mass for the mature protein of 84.2 KDa and an isoelectric point of 5.07. The highest identity with a characterized fungal enzyme, is with a ß-xylosidase from Aspergillus oryzae (70%). The optimal activity of Xyl2 is found at pH 2.0 and 28 °C. The enzyme is most stable at pH 2.0 and conserves 40% of activity at 42 °C (after 1h incubation). The kinetic parameters for p-nitrophenyl-ß-d-xylopyranoside are: KM 0.53 mM, kcat 1*107 s-1 and kcat/KM 1.9*1010 M-1 s-1. The enzyme is about 10% active on p-nitrophenyl-α-l-arabinofuranoside. Xyl2 exhibits a high hydrolytic activity on xylooligosaccharides; it liberates xylose from beechwood and birchwood glucuronoxylan and it acts synergistically with endoxylanases in the degradation of xylan. Its low pH optimum make this enzyme particularly useful in potential applications requiring a low pH such as increasing the flavor of wine.

Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteria Pseudoalteromonas sp. 2-3.

α-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of α-amylase from Pseudoalteromonas sp. 2-3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the α-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His6-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant α-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a Km of 6.94 mg/ml and Vmax of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 20 °C. This enzyme was strongly activated by Ca2+; results consistent with other α-amylases. To the best of our knowledge, this enzyme has the lowest temperature optimum so far reported for α-amylases.

Saccharification of Brown Macroalgae Using an Arsenal of Recombinant Alginate Lyases: Potential Application in the Biorefinery Process.

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6-7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20-30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.

Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291.

PURPOSE: Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. EXPERIMENTAL DESIGN: To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. RESULTS: The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. CONCLUSION: This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections.

Penicillium purpurogenum produces a novel endo-1,5-arabinanase, active on debranched arabinan, short arabinooligosaccharides and on the artificial substrate p-nitrophenyl arabinofuranoside.

Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate. The enzyme has an optimal pH of 4.5 and an optimal temperature of 30-35 °C. Calcium does not activate ABN1. ABN1 belongs to GH family 43 sub-family 6, and a Clustal alignment with sequences of characterized fungal ABNs shows highest identity (54.6%) with an ABN from Aspergillus aculeatus. A three-dimensional model of ABN1 was constructed and the docking with pNPAra was compared with similar models of an enzyme very active on this substrate and another lacking activity, both from GH family 43. Differences in the number of hydrogen bonds between enzyme and substrate, and distance between the substrate and the catalytic residues may explain the differences in activity shown by these enzymes.

Biochemical and structural characterization of Penicillium purpurogenum α-D galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition.

The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The α-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of α-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 °C, respectively. The KM for p-nitrophenyl-α-D-galactopyranoside (GalαpNP) is 0.138 mM. The enzyme is inhibited by GalαpNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an α-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications.

Heterologous expression and characterization of α-L-arabinofuranosidase 4 from Penicillium purpurogenum and comparison with the other isoenzymes produced by the fungus.

Penicillium purpurogenum secretes at least four arabinofuranosidases. In this work, the gene of α-L-arabinofuranosidase 4 (ABF4) has been sequenced and expressed in Pichia pastoris. The gene is 1521 pb long, has no introns and codes for a protein of 506 amino acid residues including a signal peptide of 26 residues. Mature protein has a calculated molecular mass of 55.4 kDa, shows 77% identity with α-L-arabinofuranosidase 1 from P. purpurogenum and belongs to family 54 of the glycosyl hydrolases. Purified enzyme has a molecular mass near 68 kDa, is active on p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl-ß-D-galactofuranoside, and follows Michaelis-Menten kinetics with KM of 1.58 ± 0.13 mM and 5.3 ± 1.18 mM, respectively. The pH optimum is 4.6 and optimal temperature is 50 °C. The enzyme is active on sugar beet arabinan and wheat flour arabinoxylan but does not act on short arabinooligosaccharides or debranched arabinan. It shows synergistic effect on arabinose liberation from wheat arabinoxylan when combined with endoxylanase from P. purpurogenum. The properties of ABF4 have been compared with those of the other arabinofuranosidases produced by the fungus. P. purpurogenum is the first fungus possessing four biochemically characterized arabinofuranosidases. The availability of four different ABFs may be valuable for biotechnological applications.

Cold-active xylanase produced by fungi associated with Antarctic marine sponges.

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.

Penicillium purpurogenum produces two GH family 43 enzymes with ß-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.

ß-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with ß-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-ß-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, ß-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3-pNPXyl, ABF3-pNPAra and XYL1-pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1-pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91,725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100,000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5-5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.

Α-L-arabinofuranosidase 3 from Penicillium purpurogenum (ABF3): potential application in the enhancement of wine flavour and heterologous expression of the enzyme.

An α-l-arabinofuranosidase (ABF3) from Penicillium purpurogenum was purified and its possible biotechnological application in the enhancement of wine flavour combined with P. purpurogenum ß-glucosidase was studied. A must from Muscat of Alexandria was used to isolate the glycosides. The total monosaccharide (glucose, arabinose and xylose) levels in the glycosides were determined after acid hydrolysis, and were compared with the result of enzymatic hydrolysis. These results were analogous to those obtained in similar experiments using a commercial preparation, thus suggesting that the enzyme from P. purpurogenum may prove useful in this particular application. This prompted us to express the enzyme heterologously. The abf3 gene was thus expressed in Pichia pastoris. The recombinant enzyme was purified and it shows the same properties of the native ABF3 (substrate specificity, kinetic constants, pH and temperature optima and antibody cross-reactivity).

Novel bifunctional alpha-L-arabinofuranosidase/xylobiohydrolase (ABF3) from Penicillium purpurogenum.

The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-D-xylopyranoside with a K(m) of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both alpha-L-arabinofuranosidase and xylobiohydrolase functionalities.

A family 51 alpha-l-arabinofuranosidase from Penicillium purpurogenum: purification, properties and amino acid sequence.

The soft rot fungus Penicillium purpurogenum secretes a wide variety of xylanolytic enzymes to the medium, among them three alpha-l-arabinofuranosidases. This work refers to arabinofuranosidase 2 (ABF 2). This enzyme was purified to homogeneity and characterized; it is a glycosylated monomer with a molecular weight of 70 000 and an isoelectric point of 5.3. When assayed with p-nitrophenyl alpha-l-arabinofuranoside (pNPAra) the enzyme followed Michaelis-Menten kinetics with a K(M) of 0.098mm. The optimum pH is 5 and the optimal temperature 60 degrees C. ABF 2 showed weak activity on natural polymeric substrates, such as sugar beet arabinan, debranched arabinan, and arabinoxylan. These results, together with its low K(M) (pNPAra) and its activity towards short arabinooligosaccharides, suggest that the enzyme belongs to the exo alpha-l-arabinosyl hydrolases not active on polymers. The abf2 gene and its cDNA were sequenced, and the gene was found to possess seven introns. The mature protein is 618 amino acids long with a calculated molecular weight of 67 212. Amino acid sequence alignments show that the enzyme belongs to family 51 of the glycosyl hydrolases, although it differs in some properties from other enzymes of this family.

Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene.

At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.