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Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans ( Tremellomycetes ) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N -glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. Key points • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Pullulan/chitosan-based multifunctional edible composite films were fabricated by reinforcing mushroom-mediated zinc oxide nanoparticles (ZnONPs) and propolis. The ZnONPs were synthesized using enoki mushroom extract and characterized using physicochemical methods. The mushroom-mediated ZnONPs showed an irregular shape with an average size of 26.7 ± 8.9 nm. The combined incorporation of ZnONPs and propolis pointedly improved the composite film’s UV-blocking property without losing transparency. The reinforcement with ZnONPs and propolis improved the mechanical strength of the pullulan/chitosan-based film by ~25%. Additionally, the water vapor barrier property and hydrophobicity of the film were slightly increased. In addition, the pullulan/chitosan-based biocomposite film exhibited good antioxidant activity due to the propolis and excellent antibacterial activity against foodborne pathogens due to the ZnONPs. The developed edible pullulan/chitosan-based film was used for pork belly packaging, and the peroxide value and total number of aerobic microorganisms were significantly reduced in meat wrapped with the pullulan/chitosan/ZnONPs/propolis film.

Edible biopolymer (pullulan/carrageenan) based functional composite films were fabricated by the addition of copper sulfide nanoparticles (CuSNP) and D-limonene (DL). The DL and CuSNP were compatible with the pullulan/carrageenan biopolymer matrix. The addition of CuSNP significantly increased the UV-blocking properties without substantially reducing the transparency of the film. The addition of CuSNP improved the film’s tensile strength by 10%; however, the DL addition did not significantly influence the strength, while the combined addition of CuSNP and DL increased the strength by 15%. The addition of the fillers did not significantly affect the thermal stability of the film, but the water vapor barrier property was slightly improved. There was no significant change in the moisture content and hydrophobicity of the composite film. Besides, the composite film showed some antimicrobial activity against food-borne pathogenic bacteria. The fabricated pullulan/carrageenan-based film with antimicrobial and UV-barrier properties is likely to be used in active food packaging applications.

Background Aureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans. Results The 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus. Conclusions The differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans , A. melanogenum , A. subglaciale and A. namibiae . The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.

Thermosensitive chitosan/β-glycerophosphate (CS/BGP) systems have been developed as injectable hydrogels. However, the hydrogels exhibited poor mechanical properties due to their physically crosslinked networks. In this work, CS/BGP hydrogels were reinforced by covalent crosslinking using genipin (GE) and concomitantly semi-interpenetrating networks using pullulan (PL). Based on response surface methodology, the optimized formulation was composed of CS (1.05%, w/v), PL (1%, w/v), BGP (6%, w/v), and GE (70.79 mcg/mL). The optimized hydrogels exhibited Young’s modulus of 92.65 ± 4.13 kPa and a percentage of equilibrium swelling ratio of 3259.09% ± 58.90%. Scanning electron micrographs revealed a highly porous structure with nanofibrous networks in the CS/PL/BGP/GE hydrogels. The chemical interactions between the compositions were investigated by Fourier-transform infrared spectroscopy. Rheological measurements illustrated that the optimized hydrogels displayed sol–gel transition within one minute at 37 °C, a lower critical solution temperature of about 31 °C, and viscoelastic behavior with high storage modulus. Furthermore, the optimized hydrogels demonstrated higher resistance to in vitro enzymatic degradation, compared to the hydrogels without GE. Our findings could suggest that the thermosensitive CS/PL/BGP/GE hydrogels with enhanced mechanical properties and swelling capacity demonstrate the potential for use as scaffolds and carriers for cartilage tissue engineering and drug delivery applications.

α-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12–14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains (GH13_41) in two-domain amylase–pullulanases. GH57 harbours type II pullulanases. Specificity differences, domain organisation, carbohydrate binding modules, sequence motifs, three-dimensional structures and specificity determinants are discussed. The phylogenetic analysis indicated that GH13_39 enzymes could represent a “missing link” between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference.

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan's chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed. [PUBLICATION ABSTRACT]

Wound healing is a complex process of overlapping phases with the primary aim of the creation of new tissues and restoring their anatomical functions. Wound dressings are fabricated to protect the wound and accelerate the healing process. Biomaterials used to design dressing of wounds could be natural or synthetic as well as the combination of both materials. Polysaccharide polymers have been used to fabricate wound dressings. The applications of biopolymers, such as chitin, gelatin, pullulan, and chitosan, have greatly expanded in the biomedical field due to their non-toxic, antibacterial, biocompatible, hemostatic, and nonimmunogenic properties. Most of these polymers have been used in the form of foams, films, sponges, and fibers in drug carrier devices, skin tissue scaffolds, and wound dressings. Currently, special focus has been directed towards the fabrication of wound dressings based on synthesized hydrogels using natural polymers. The high-water retention capacity of hydrogels makes them potent candidates for wound dressings as they provide a moist environment in the wound and remove excess wound fluid, thereby accelerating wound healing. The incorporation of pullulan with different, naturally occurring polymers, such as chitosan, in wound dressings is currently attracting much attention due to the antimicrobial, antioxidant and nonimmunogenic properties. Despite the valuable properties of pullulan, it also has some limitations, such as poor mechanical properties and high cost. However, these properties are improved by blending it with different polymers. Additionally, more investigations are required to obtain pullulan derivatives with suitable properties in high quality wound dressings and tissue engineering applications. This review summarizes the properties and wound dressing applications of naturally occurring pullulan, then examines it in combination with other biocompatible polymers, such chitosan and gelatin, and discusses the facile approaches for oxidative modification of pullulan.

After over 100 strains of Aureobasidium spp. isolated from mangrove system were screened for their ability to produce exopolysaccharide (EPS), it was found that Aureobasidium pullulans var. melanogenium P16 strain among them could produce high level of EPS. Under the optimal conditions, 65.3 g/L EPS was produced by the P16 strain within 120 h at flask level. During 10-L batch fermentation, when the medium contained 120.0 g/L sucrose, 67.4 g/L of EPS and 23.1 g/L of cell dry weight in the culture were obtained within 120 h, leaving 0.78 g/L of reducing sugar and 11.4 g/L of total sugar in the fermented medium. It should be stressed that during the fermentation, no melanin was observed. After purification, the purified EPS was confirmed to be pullulan. This is the first time to report that A. pullulans var. melanogenium P16 strain isolated from the mangrove system can produce high level of pullulan.

The present opinion deals with the re‐evaluation of pullulan (E 1204) when used as a food additive and with the new application on the extension of use to several food categories. Pullulan (E 1204) is obtained by fermentation of a food‐grade hydrolysed starch with non‐genetically modified Aureobasidium pullulans ■■■■■. Based on the available information, the Panel considered that the manufacturing process of pullulan (E 1204) using this microorganism does not raise a safety concern. The Panel confirmed that pullulan (E 1204) is of no concern for genotoxicity. In vitro, pullulan (E 1204) is broken down by salivary and pancreatic amylase and intestinal iso‐amylase and it is further metabolised to short chain fatty acids in the colon by fermentation. Human adult volunteer studies suggested that effects of pullulan (E 1204) are similar to the effects of other poorly digestible carbohydrate polymers including modified celluloses and that mild undesirable gastrointestinal symptoms (i.e. abdominal fullness, flatulence, bloating and cramping) may occur at doses of 10 g pullulan per day and greater. The Panel compared the dose of 10 g pullulan per day with the dietary exposure estimates to pullulan (E 1204) in its currently permitted uses and considering the proposed changes to the currently permitted uses. The Panel concluded that there is no need for a numerical ADI for pullulan (E 1204) and there is no safety concern for the currently reported uses and use levels. Additionally, the Panel concluded that the exposure estimates considering the proposed changes to the currently permitted uses and use levels of pullulan (E 1204) are of no safety concern. The estimates for dietary exposure to pullulan (E 1204) indicate that individuals with a high level of exposure, principally coming from food supplements, may experience mild gastrointestinal symptoms at the currently reported uses and use levels.