Analysis of the molecular basis for the non-amylolytic and non-proteolytic nature of Aspergillus vadensis CBS 113365.

Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.

Validation of the Test Method-Determination of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Related Food Products and Animal Feeds: Collaborative Study, Final Action 2020.07.

BACKGROUND: A simple, accurate, and reliable method to measure available carbohydrate components of food products, including cereal and dairy products, fruits, vegetables, processed food, food ingredients, and animal foods, was developed by Megazyme (product K-AVCHO, Bray, Ireland). A single-laboratory validation of the enzymatic method resulted in First Action status as Official Method of AnalysisSM2020.07. OBJECTIVE: A collaborative study was conducted to evaluate the repeatability and reproducibility of Official Method 2020.07 for the measurement of available carbohydrates, including digestible starch, lactose, sucrose, isomaltose, maltose, glucose, fructose, and galactose in a broad range of food and feed products. METHOD: Samples are defatted if containing >10% fat content, and incubated with pancreatic α-amylase and amyloglucosidase under conditions that simulate those in the small intestine (pH 6, 37°C, 4 h). The reaction solution is clarified and diluted, and an aliquot is incubated with sucrase, maltase, oligo-1,6-α-glucosidase, and ß-galactosidase to hydrolyze sucrose, maltose, isomaltose, and lactose to glucose, fructose, and galactose, which are then measured enzymatically. The multi-laboratory validation (MLV) matrixes included cereal, animal feeds, fruit, vegetables, infant formula, powdered milk drink, a dessert product, and mushrooms. Additional materials were analyzed by collaborators as "practice samples." RESULTS: All MLV matrixes resulted in repeatability relative standard deviations (RSDr) <3.91% and reproducibility relative standard deviations (RSDR) ranging from 3.51 to 11.58% with 9 of the 10 matrixes having RSDR of <6.19%. For the practice samples, the RSDR ranged from 2.7 to 11.4% with 7 of the 8 samples having RSDR of <4.4%. CONCLUSIONS: Official Method 2020.07 meets the AOAC requirements for repeatability and reproducibility, and the data support Final Action status. HIGHLIGHTS: Official Method 2020.07 is a robust, simple to use, and reproducible method for the analysis of available carbohydrates in a wide range of matrixes.

Determination of Fructan (Inulin, FOS, Levan, and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients): Single-Laboratory Validation, First Action 2018.07.

Traditional enzyme-based methods for measurement of fructan were designed to measure just inulin and branched-type (agave) fructans. The enzymes employed, namely exo-inulinase and endo-inulinase, give incompletely hydrolysis of levan. Levan hydrolysis requires a third enzyme, endo-levanase. This paper describes a method and commercial test kit (Megazyme Fructan Assay Kit) for the determination of all types of fructan (inulin, levan, and branched) in a variety of animal feeds and pet foods. The method has been validated in a single laboratory for analysis of pure inulin, agave fructan, levan, and a range of fructan containing samples. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo-inulinase, endo-inulinase, and endo-levanase, followed by measurement of these sugars using the PAHBAH reducing sugar method which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are specifically hydrolyzed and removed by borohydride reduction. The single-laboratory validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit®), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX®) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included working range, target selectivity, recovery, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. The method is robust, quick, and simple.

A novel enzymatic method for the measurement of lactose in lactose-free products.

BACKGROUND: In recent years there has been a surge in the number of commercially available lactose-free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono-, di-, and oligosaccharides. RESULTS: In the current work, we describe the development of a novel enzymatic low-lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre-treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a ß-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a 'creep' calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose-free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose-free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC-PAD) analysis. CONCLUSION: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose-free samples. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Novel substrates for the automated and manual assay of endo-1,4-ß-xylanase.

endo-1,4-ß-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

A novel automatable enzyme-coupled colorimetric assay for endo-1,4-ß-glucanase (cellulase).

endo-1,4-ß-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-D-cellopentaoside. Graphical Abstract Principle of the CELLG5 assay.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by ß-xylanase, α-L-arabinofuranosidase and ß-xylosidase.

A range of α-L-arabinofuranosyl-(1-4)-ß-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 ß-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and ß-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum ß-xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3(2)-α-L-Araf-(1-4)-ß-D-xylobiose (A(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(3)XX), 2(2)-α-L-Araf-(1-4)-ß-D-xylotriose (XA(2)X), 3(2)-α-L-Araf (1-4)-ß-D-xylotriose (XA(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotriose (A(2+3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2+3)XX), 2(4),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(2+3)XXX) and 3(3),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(3)A(3)XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A(2,3)XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with ß-xylosidase and ß-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

Overexpression, purification and characterisation of homologous α-L-arabinofuranosidase and endo-1,4-ß-D-glucanase in Aspergillus vadensis.

In the recent past, much research has been applied to the development of Aspergillus, most notably A. niger and A. oryzae, as hosts for recombinant protein production. In this study, the potential of another species, Aspergillus vadensis, was examined. The full length gDNA encoding two plant biomass degrading enzymes, i.e. α-L-arabinofuranosidase (abfB) (GH54) and endo-1,4-ß-D-glucanase (eglA) (GH12) from A. vadensis were successfully expressed using the gpdA promoter from A. vadensis. Both enzymes were produced extracellularly in A. vadensis as soluble proteins and successfully purified by affinity chromatography. The effect of culture conditions on the expression of abfB in A. vadensis was examined and optimised to give a yield of 30 mg/L when grown on a complex carbon source such as wheat bran. Characterization of the purified α-L-arabinofuranosidase from A. vadensis showed an optimum pH and temperature of pH 3.5 and 60 °C which concur with those previously reported for A. niger AbfB. Comparative analysis to A. niger AbfA demonstrated interesting differences in temperate optima, pH stability and substrate specificities. The endo-1,4-ß-D-glucanase from A. vadensis exhibited a pH and temperature optimum of pH 4.5 and 50 °C, respectively. Comparative biochemical analysis to the orthologous EglA from A. niger presented similar pH and substrate specificity profiles. However, significant differences in temperature optima and stability were noted.

Colourimetric and fluorometric substrates for measurement of pullulanase activity.

Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and ß-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-ß-maltotriosyl (1-6) α-maltotrioside (BzCNPG3G3, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-ß-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, ß-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end D-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-ß-maltotrioside (3) or 4-methylumbelliferyl-ß-maltotrioside (4) liberated is immediately hydrolysed to D-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.

Physiological and molecular aspects of degradation of plant polysaccharides by fungi: what have we learned from Aspergillus?

Plant biomass is the most abundant and usable carbon source for many fungal species. Due to its diverse and complex structure, fungi need to produce a large range of enzymes to degrade these polysaccharides into monomeric components. The fine-tuned production of such diverse enzyme sets requires control through a set of transcriptional regulators. Aspergillus has a strong potential for degrading biomass, thus this genus has become the most widely studied group of filamentous fungi in this area. This review examines Aspergillus as a successful degrader of plant polysaccharides, and reviews its potential in many industries such as biofuel and as a production host of homologous and heterologous proteins.

Measurement of endo-1,4-ß-glucanase.

Several procedures are available for the measurement of endo-1,4-ß-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.

Promiscuity in ligand-binding: The three-dimensional structure of a Piromyces carbohydrate-binding module, CBM29-2, in complex with cello- and mannohexaose.

Carbohydrate-protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, "carbohydrate-binding modules" (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. "CBM29" modules, derived from a noncatalytic component of the Piromyces equi cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both beta-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a beta-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.

Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold.

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the alpha-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 A resolution, reveals a five-bladed beta-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially enclosed at one end, forms a single extended substrate-binding surface across the face of the propeller. Three carboxylates deep in the active site groove provide the general acid and base components for glycosidic bond hydrolysis with inversion of anomeric configuration.