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.

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.

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.

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.

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.