Search

Polymalate (PMA) produced by the whole genome duplicated strain Aureobasidium melanogenum OUC had a high molecular weight (Mw) of 3.9 × 10

Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of (NH4)2SO4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreAS628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreAS628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp.

Bone defect is usually difficult to recover quickly, and bone scaffold transplantation is considered to be an effective method. Biomaterials have a wide range of application prospects in bone tissue repair, and the two key problems are the selection of materials and cells. The object of this study was to discuss the structural characteristics of bone scaffold materials and their effects on bone repair in vivo. The chitin-hydroxyapatite (HAP)-collagen composite scaffolds (CHCS) was prepared with epichlorohydrin (ECH) as crosslinking agent. The structure was characterized and the compressive strength, porosity, water absorbency and stability were investigated. The biocompatibility and osteogenic differentiation of CHCS in vitro were detected, and the effect of defect repair in vivo was evaluated. The results suggested that HAP not only enhanced the compressive strength of CHCS, but also promoted the formation of calcium nodules due to its bone conductivity. Histological staining showed that collagen promoted collagen deposition and new bone formation. X-ray images also indicated that CHCS transplantation accelerated bone repair. Therefore, CHCs has immense potential in bone regeneration.

Several dressing materials can be used efficiently in recent times, both in their natural and synthetic combinations like; microfibers, film, nanofibers, hydrogels, and various drugs. The specific characteristics, such as biocompatibility and providing a favorable environment for wound healing, make many polysaccharides pivotal as wound dressings. Keeping in view the importance of these polysaccharides, we have developed novel chitosan-ulvan hydrogel incorporated by cellulose nanocrystals (CNCs) loading epidermal growth factor (EGF) drug (CS-U-CNC-EGF) by the freeze-dried process. The morphological features of novel hydrogel were perceived by FTIR, XRD, FESEM, and DSC analysis. The incorporation of the nanocrystals content modified the porous microstructure at pore size from 237 ± 59 μm to 53 ± 16 μm, improved mechanical stress curve from 0.57 MPa to 1.2 MPa, thermal and swelling behavior. The novel nanocomposites revealed non-toxic behavior and excellent cell proliferation. Whereas hydrogel showed sustained release of the epidermal growth factor (EGF), thereby enhancing EGF delivery at the wound site for 15 days from a 100% wound contraction treated group. Moreover, the controlled release of EGF from CS-U-CNC-EGF hydrogels showed significantly faster-wound healing efficiency concerning considerably faster granulations tissue formation and collagen deposition. The study's results point to possible future applications of this composite hydrogel in wound healing as a wound dressing material.

Hydrophobic drug encapsulation inside targeted nanoparticles can enhance accumulation in inflamed sites, limit toxicity to healthy tissue, and improve pharmacokinetics compared to free drug dosing. This study reports a functionalized marine polysaccharide nanoparticle with a controlled release, targeting abilities, and in-situ imaging properties. Carbon dots functionalized Enteromorpha polysaccharide/Mannose/Methionine functionalized Chitosan (CDs.EP/Man/Meth.Cs) NPs could deliver apremilast to inflammatory macrophages and Caco-2 intestinal cells as an in vitro model for application in oral drug delivery to cure IBD. The nanoparticles were simply a polyelectrolyte complex between cationic functionalized chitosan and anionic polysaccharide of Enteromorpha prolifera. Functionalized polysaccharides and the prepared NPs were well characterized. The functionalized nanoparticles could overcome the limitation of poor drug bioavailability and showed a high loading capacity of (45 %) with a controlled release of about (74.5 %). Confocal laser scanning imaging showed higher cellular uptake of the modified nanoparticles than that of the unmodified nanoparticles in LPS-activated RAW 264.7 macrophages and Caco-2 cells. The effect of functionalization on the cellular uptake targetability was assessed using spectrofluorometric measurements after mannose competition. Anti-inflammatory activity of apremilast-loaded NPs is more elevated than the free drug. These results suggest the feasibility of using functionalized EP/Cs nanoparticles in IBD oral drug delivery.

It has been well documented that different strains of Aureobasidium spp. can synthesize and secrete over 30.0 g/L of polymalate (PMA) and the produced PMA has many potential applications in biomaterial, medical and food industries. The substrates for PMA biosynthesis include glucose, xylose, fructose, sucrose and glucose or fructose or xylose or sucrose-containing natural materials from industrial and agricultural wastes. Malate, the only monomer for PMA biosynthesis mainly comes from TCA cycle, cytosolic reduction TCA pathway and the glyoxylate cycle. The PMA synthetase (a NRPS) containing A like domain, T domain and C like domain is responsible for polymerization of malate into PMA molecules by formation of ester bonds between malates. PMA biosynthesis is regulated by the transcriptional activator Crz1 from Ca2+ signaling pathway, the GATA-type transcription factor Gat1 from nitrogen catabolite repression and the GATA-type transcription factor NsdD.

In order to increase content of glucuronic acid in the exopolysaccharide (EPS) and its flocculating activity, an UDP-glucose dehydrogenase gene was overexpressed in Lipomyces starkeyi V19. The obtained U9 strain could produce 62.1 ± 1.2 g/l EPS while the V19 strain only produced 53.5 ± 1.3 g/l EPS. The compositions of monosaccharides (mannose, glucuronic acid and galactose) in the purified EPS (U9-EPS) from the U9 strain contained 3.79:1:5.52 while those in the purified EPS (V19-EPS) were 3.94:1:6.29. The flocculation rate of the U9-EPS on kaolin clay reached 87.9%, which was significantly higher than that (74.7%) of the V19-EPS while the decolorization rate of Congo Red (CR) by the U9-EPS reached 94.3%, which was significantly higher than that of CR by the V19-EPS (86.23%). The results showed that the purified bioflocculant U9-EPS had effective flocculation of kaolin clay. The U9-EPS also had high ability to flocculate the polluted river water and decolorize Congo red.

In our previous study, it was found that Aureobasidium melanogenum TN3-1 was a high pullulan producing and osmotic tolerant yeast-like fungal strain. In this study, the HOG1 signaling pathway controlling glycerol synthesis, glycerol, trehalose and vacuoles were found to be closely related to its pullulan biosynthesis and high osmotic tolerance. Therefore, deletion of the key genes for the HOG1 signaling pathway, glycerol and trehalose biosynthesis and vacuole formation made all the mutants reduce pullulan biosynthesis and increase sensitivity of the growth of the mutants to high glucose concentration. Especially, abolishment of both the VSP11 and VSP12 genes which controlled the fission/fusion balance of vacuoles could cause big reduction in pullulan production (less than 7.4 ± 0.4 g/L) by the double mutant ΔDV-5 and increased sensitivity to high concentration glucose, while expression of the VSP11 gene in the double mutant ΔDV-5 made the transformants EV-2 restore pullulan production and tolerance to high concentration glucose. But cell growth of them were the similar. The double mutant ΔDV-5 had much bigger vacuoles and less numbers of vacuoles than the transformant EV-2 and its wild type strain TN3-1 while it grew weakly on the plate with 40% (w/v) glucose while the transformant EV-2 and its wild type strain TN3-1 could grow normally on the plate even with 60% (w/v) glucose. The double mutant ΔDV-5 also had high level of pigment and its cells were swollen. This was the first time to give the evidence that glycerol, trehalose and vacuoles were closely related to pullulan biosynthesis and high osmotic tolerance by A. melanogenum.

The fungal α-glucan synthases (Agss) are multi-domain proteins catalyzing biosynthesis of cell wall α-1,3-glucan which determines cell wall integrity or fungal pathogenicity and pullulan which is a maltotriosyl polymer made of α-1,4 and α-1,6 bound glucose units. The Agss family can be divided into 11 groups, some of which lost the original functions due to accumulation of harmful mutations or gene loss. Schizosaccharomyces pombe kept five kinds of Agss in the genome while Aspergillus spp. and Penicillium spp. lost one or two or three kinds of Agss. All the human, animal and plant pathogens kept only one single kind of Ags or only one active Ags for synthesis of cell wall α-1,3-glucan, a virulence factor. While the genus Aureobasidium spp. contained three kinds of Agss, of which only some of the Ags2 was involved in pullulan biosynthesis. Although many Agss contained Big_5 domain, only the Big_5 domain with conserved amino acids LQS from some strains of A. melanogenum could catalyze pullulan biosynthesis. This whole amino acid sequence and phylogenetic differences may cause non-α-1,3-glucan synthesizing activity of some fungal Agss.

Pullulan is an important polysaccharide. Although its synthetic pathway in Aureobasidium melanogenum has been elucidated, the mechanism underlying its biosynthesis as regulated by signaling pathway and transcriptional regulator is still unknown. In this study, it was found that the expression of the UGP1 gene encoding UDPG-pyrophosphorylase (Ugp1) and other genes which were involved in pullulan biosynthesis was controlled by the transcriptional activator Msn2 in the nuclei of yeast-like fungal cells. The Ugp1 was a rate-limiting enzyme for pullulan biosynthesis. In addition, the activity and subcellular localization of the Msn2 were regulated only by the cAMP-PKA signaling pathway. When the cAMP-PKA activity was low, the Msn2 was localized in the nuclei, the UGP1 gene was highly expressed, and pullulan was actively synthesized. By contrast, when the cAMP-PKA activity was high, the Msn2 was localized in the cytoplasm and the UGP1 gene expression was disabled so that pullulan was stopped, but lipid biosynthesis was actively enhanced. This study was the first to report that pullulan and lipid biosynthesis in yeast-like fungal cells were regulated by the Msn2 and cAMP-PKA signaling pathway. Elucidating the regulation mechanisms was important to understand their functions and enhance pullulan and lipid biosynthesis.

Although pullulan has many uses in industry, the detailed mechanisms of its biosynthesis still require clarification. In this study, it was found that a short α-1,4-glucosyl chain (pullulan primer) synthesized by the glycogenins Glg1 and Glg2 for initiation of glycogen biosynthesis was also needed for pullulan synthesis. The primers were also synthesized on sterol glycosides and glucosylceramides by catalysis of sterol glucosyltransferase (Sgt1) and ceramide β-glucosyltransferase (Gcs1). All the primers might be elongated to be long α-1,4-glucosyl chain (pullulan precursor) by catalysis of the glycogen synthetase domain of the AmAgs2 as previously reported. Then, the amylase domain of the same AmAgs2 was responsible for pullulan biosynthesis. Removal of all the genes encoding Glg1, Glg2, Gcs1 and Sgt1 made all the mutants produce much less pullulan than the strain P16. However, pullulan synthesis could not be stopped totally in these mutants, suggesting that any other unknown alternative pullulan primers may exist in the yeast cells. Complementation of all the genes in the mutants restored pullulan biosynthesis. This is the first time to report that like starch and glycogen biosynthesis, alternative primers are also required for pullulan biosynthesis in Aureobasidium melanogenum P16.

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.

The biomass from Enteromorpha prolifera is used to exploit and transfer this common green bloom algae into new value-added products. In this work, cellulose nanocrystals (CNCs) were first prepared with the cellulose extracted from the biomass of E. prolifera. Initially, cellulose was treated by alkali and bleaching treatments. Subsequently, CNCs were isolated by acid hydrolysis with different concentrations. Structural changes and crystallinity were characterized by Fourier transform infrared spectroscopy (FTIR) spectroscopy, X-ray diffraction (XRD) analysis and t thermogravimetric analysis. The crystallinity index and crystallite size of the prepared CNCs depended mainly on acid concentrations. TEM observation revealed that the CNCs obtained by hydrolysis with 60% acid (CNC60) exhibited a ‘needle-like’ nanocrystals with a length of 177 ± 12 nm and width of 3 ± 1 nm, indicating that the CNC60 had a high ratio aspect and could serve as reinforcement nanofiller. Further investigation showcased that the addition of CNC60 exhibited the best effect for enhancing the mechanical properties of bionanocomposite films derived from the water-soluble polysaccharides extracted from the biomass of E. prolifera. Therefore, a new strategy was offered in this study for the comprehensive utilization of E. prolifera biomass to fabricate them into high-strength film materials.

A novel yeast strain Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert was found to be able to produce a high level of extracellular polysaccharide (EPS). Under the optimal conditions, the yeast strain could yield 73.25 ± 2.3 g/L of EPS within 5 days at a flask level. During a 10-liter fermentation, the yeast strain could produce 78.05 ± 3.5 g/L of EPS within 120 h. The FT-IR spectra of the standard pullulan from Sigma and the purified EPS produced by A. melanogenum 13-2 were almost identical and the purified EPS could be actively hydrolyzed by a pullulanase, demonstrating that the purified EPS was pullulan. The molecular weight (Mw) of the purified pullulan was estimated to be 7.703 × 105 g/moL. Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 lose the ability to synthesize any pullulan. The mutant DAG27 was more sensitive to radiation of UV light, high NaCl concentration, heat treatment, strong oxidation of H2O2 and desiccation than its wild type strain 13-2, suggesting that the produced pullulan could play an important role in resistance of the yeast cells to various stresses. This was the first time to show that the yeast strain obtained from the desert could produce such high level pullulan and the produced pullulan had an obviously protective effect on its producer.

In the present experimental series, we have developed a novel nanocomposite to target activated macrophages in the colon with real time imaging and therapeutic capabilities. This binary nanocomposite was formed by the covalent conjugation of mannosylated NPs (Man-NPs) with carbon dots (CDs). Man-NPs were prepared using a self-assembly method based on mannosylated decamethylenediamine-grafted carboxymethyl inulin amphiphilic acid. While, the CDs were synthesized using a simple bottom-up process using citric acid monohydrate and diethylenetriamine, which were tightly bonded to the Man-NPs surface by carbodimide coupling. The resulting nanocomposite had a uniform size of 241.3 nm with a negative charge and a high drug casing density of 25.54 wt% and blue self-fluorescence were emitted. Whereas, in vitro observation of cellular uptake indicated the greater nanocomposite uptake in inflamed macrophage as compared to the untreated macrophage and mannose receptor-negative cell lines, 4T1 respectively. However, in vivo bio distribution exhibited a large number (60%) of CDs/Man-NPs nanocomposite accumulated in the inflamed colon of colitis mice. It should be noted that the novel nanocomposite, as macrophage-targeted drug delivery, could have promise for the treatment of inflammatory bowel disease (IBD).

A purified polysaccharide from Enteromorpha prolifera (PEP) with a molecular mass of 147.8kDa, was prepared by hot-water extraction method and purified by anion-exchange chromatography and gel filtration chromatography. Low Mw polysaccharide of E. prolifera (LEP, 44.8kDa) was obtained by enzymatic degradation. Sulfated derivatives of the two different Mw polysaccharides were synthesized by chlorosulfonic acid/pyridine method, and the degrees of substitution of SPEP and SLEP were 0.57 and 0.81, respectively. Physicochemical properties and FT-IR spectra showed that enzymatic degradation and sulfated modification of polysaccharides were successful. 13C NMR analysis showed that the sulfate groups mainly attached to C-6 of rhamnose. Moreover, the study revealed that enzymatic degradation and sulfated modification of polysaccharides improved significantly superoxide, hydroxyl and DPPH radicals scavenging activities in vitro, and enhanced obviously moisture absorption/retention capacities. Therefore, these results demonstrated that molecular weight and sulfate groups had obvious effects on antioxidant activities and moisture absorption/retention abilities of the polysaccharides from E. prolifera.

After the compositional change of a pullulan production medium, a molecular weight (Mw) of the pullulan produced by Aureobasidium melanogenum P16 was 2.32 × 10 6 and a pullulan titer was 44.4 g/L while a Mw of the pullulan produced by A. melanogenum P16 grown in the initial medium was only 3.47 × 10 5 and a pullulan titer was 65.3 g/L. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase while the decreased pullulan titer was related to the decreased transcriptional levels of the genes encoding 6-P-glucose kinase, glucosyltransferase, α-phosphoglucose mutase, UDPG-pyrophosphorylase and pullulan synthetase. During the 10-L fermentation, when the yeast strain P16 was grown in the initial medium, the pullulan and oligosaccharide titers were 65.5 g/L and 7.8 g/L, respectively and the Mw of the produced pullulan was 4.42 × 10 5 while when the yeast strain P16 was grown in the compositionally changed medium, the pullulan and oligosaccharide titers were 46.4 g/L and 27.8 g/L, respectively and the Mw of the produced pullulan was 2.6 × 10 6 . Most of the oligosaccharides produced by the yeast strain P16 cultivated in the compositionally changed medium had degree of polymerization of 4 and 5. Therefore, both of the high Mw pullulan and oligosaccharides with high levels were produced by the yeast strain P16.

It has been well known that poly(β-l-malic acid)(PMA) has many potential applications. However, it is still completely unknown how PMA is biosynthesized in Aureobasidium spp. In this study, it was found that malic acid from TCA cycle was the main source for PMA biosynthesis. Especially, the novel PMA synthetase encoded by the PMAs gene, a non-ribosomal peptide synthetase (NRPS) containing A like domain, T domain and C like domain was the key enzyme for polymerization of malate into PMA. Therefore, abolishment of the PMAs gene encoding the novel PMA synthetase rendered the mutant ΔPMAs-3 totally to lose the ability to synthesize any PMA and complementation of the PMAs gene partially restored PMA biosynthesis, but the mutant could grow normally on the YPD plate and in the PMA medium with CaCO3. The transcriptional activator Crz1 in the Ca2+-signal pathway controlled expression of the PMAs gene and PMA biosynthesis. The complete elucidation of the PMA biosynthesis pathway and its regulation was of significant for a deeper understanding of detailed yeast-like fungal PMA synthesis, metabolic engineering and molecular editing for modifying PMA biosynthesis and its physicochemical properties.

Pullulan, a biological macromolecule, has many applications. However, it is completely unknown how and where it is synthesized. In this study, it was found that the multidomain AmAgs2 (α-glucan synthase 2) encoded by an AmAGS2 gene in Aureobasidium melanogenum P16 contained the amylase domain (Amy_D), the glycogen synthetase domain (Gys_D) and the transmembrane regions in which the exopolysaccharide transporter domain (EPST_D) was embedded. Removal of the AmAGS2 gene in A. melanogenum P16 rendered the disruptants not to synthesize any pullulan and complementation of the AmAGS2 gene in the disruptants restored pullulan synthesis. Overexpression of the gene in Aureobasidium melanogenum CBS105.22, a non-pullulan producer, resulted in the transformants producing pullulan. Therefore, the AmAGS2 gene was the key gene responsible for pullulan biosynthesis in A. melanogenum P16. It was speculated that the short α-1,4-glucosyl chains (pullulan primers) were elongated by the Gys_D of the AmAgs2 to form long α-1,4-glucosyl chains (precursors of pullulan). All the precursors were transported to outside plasma membrane by the EPST_D in the transmembrane regions of the AmAgs2. Then, the Amy_D of the AmAgs2 was responsible for both hydrolysis of the endo-α-1,4-linkages in the precursors to release maltotriose and transfer of the maltotriose to Lph-glucose to form α-1,6 glucosidic bonds between maltotrioses in pullulan molecule. This is the first time to report that the AmAgs2 can play the key role in pullulan biosynthesis.

Overexpression of the optimized Vitreoscilla hemoglobin (VHb) gene and the native flavohemoglobin (FHb) gene in Aureobasidium melanogenum P16 rendered a V6 strain and a F44 strain to overproduce pullulan compared to that produced by their wild type strain P16. The capacity to bind CO and oxygen in the V6 strain and the F44 strain was also obviously enhanced. At the same time, the transcriptional levels of the relevant genes were also increased in the V6 strain and the F44 strain and the fused vgbop + the gene encoding GFP and FHb gene + the gene encoding GFP were also actively expressed. During a 10-liter fermentation, the P16 strain produced only 72.0 ± 1.0 g/L pullulan, the yield was 0.77 g/g of sucrose, the productivity was 0.5 ± 0.01 g/L/h and only 79.4% of the total sugar was used. In contrast, the strain V6 yielded 102.3 ± 1.8 g/L of pullulan, the yield was 0.89 g/g of sucrose, the productivity was 0.7 ± 0.01 g/L/h and 96.0% of total sugar was used while 101.4 ± 2.9 g/L of pullulan was accumulated in the culture of the strain F44, the yield was 0.88 g/g of sucrose, the productivity was 0.7 ± 0.02 g/L/h and 96.4% of total sugar was utilized. These data strongly demonstrated that the concentration of pullulan, yield, productivity and sugar utilization were greatly enhanced by overexpression of the VHb and FHb. But their cell growth was almost the similar.

The effect of antibiotics in the stomach for curing Helicobacter pylori infection is hampered by the adverse gastric environment and low bioavailability of the administered drugs. Concerning these challenges, a polymeric nano-micelle was developed. Initially, carboxymethyl chitosan (CMCS) was hydrophobically modified with stearic acid (SA), and the obtained CMCS-g-SA co-polymers was further conjugated with urea to acquire U-CMCS-g-SA co-polymers. Sphere-shaped nano-micelles (UCS-NMs) with the particle sizes of approximately 200nm were obtained with the U-CMCS-g-SA co-polymers. It was specified that this nano-micelle had no cell toxicity to AGS cells, and it could maintain a stable particle size for 6h in simulated gastric fluid and for 24h in 1×PBS. Attractively, the CMCS backbones granted this nano-micelle an excellent retention time in the stomach, almost 24h; meanwhile, the grafted ureido groups conferred effective targeting to H. pylori. This nano-micelle could load clarithromycin with high efficiency and exhibited slow release of this antibiotic in a slightly alkaline environment. In vitro inhibitory assay also indicated that a significantly enhanced anti-H. pylori activity was achieved by using this nano-micelle. This work demonstrated that the U-CMCS-g-SA nano-micelle is a proper carrier for targeted delivery of clarithromycin to H. pylori under the gastric mucus layer.

Aureobasidium melanogenum 11–1 was found to be able to produce over 281.7 ± 7.1 U/mL of β-fructofuranosidase activity. The protein deduced from the cloned β-fructofuranosidase1 gene had the conserved motif A (IGDP), motif D (RDP) and motif E (ET) and 11 N-glycosylation sites, indicating it was a β-fructofuranosidase with the high-level fructooligosaccharide (FOS) biosynthesis. Overexpression of the β-fructofuranosidase1 gene in the yeast strain 11–1 made a tranformant 33 produce 557.7 U/mL of β-fructofuranosidase activity. The molecular weight of the β-fructofuranosidase1 in which all the carbohydrates were removed by the Endo-H was 82.4 kDa. Within 7 h of the transfructosylation reaction, the yield of FOS was 0.66 g of FOS/g of sucrose and percentages of GF2, GF3 and GF4 were 79.5%, 18.9% and 1.6%. This demonstrated that the β-fructofuranosidase1 and the transformant 33 had highly potential applications in biotechnology for FOS production.

It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by β- d -fructofuranosidase were beneficial for their cell growth and pullulan biosynthesis. However, it is still unknown about how β- d -fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding β- d -fructofuranosidase1, β- d -fructofuranosidase2 and β- d -fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three β- d -fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ± 0.4 g/L), less β- d -fructofuranosidase activity (1.1 ± 0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ± 2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding β- d -fructofuranosidase1 and β- d -fructofuranosidase3 resulted in a double mutant DF4–7 producing 77.5 ± 3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ± 0.0 U/mL of β- d -fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ± 3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ± 0.0 U/mL of β- d -fructofuranosidase activity and 6.2 ± 0.5 g/L of FOSs. These confirmed that high β- d -fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the β- d -fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, β- d -fructofuranosidase1 gene and β- d -fructofuranosidase3 gene enabled the corresponding transformants to restore β- d -fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan.

Polyelectrolyte complex nanoparticles were fabricated via electrostatic complexation between Enteromorpha prolifera polysaccharide (EP) and chitosan (CS). The monodisperse EP/CS NPs with negatively charge were prepared at pH 4.0 and mixing ratio of 3.0:1.0. Curcumin loaded nanoparticles (CUR-NPs) showed spherical morphology with negatively charge of −16.27 ± 0.97 mV and average diameter range of 230 to 330 nm. The results of spectroscopic analysis, XRD and DSC confirmed that there were hydrogen-bonding interaction and hydrophobic interaction between curcumin and EP/CS NPs. The CUR-NPs improved storage, thermal and photo stability of curcumin and exhibited sustained release of curcumin in vitro. Moreover, the CUR-NPs showed higher cellular uptake than free CUR with incubation for 3 h by CLSM visualization and fluorescence quantitative assay. Furthermore, MTT assay results demonstrated that the CUR-NPs possessed good anticancer activity against B16F10 cells. Therefore, the EP-based nanoparticles are promising candidates for carriers in controllable hydrophobic anti-tumor drug delivery.

A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 106 Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ± 3.5 g/L of pullulan with a Mw of 0.35 × 106 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.

In this study, one of the glucosyltransferase genes for pullulan production was cloned from Aureobasidum melanogenum P16 and charaterized. It was found that the UGT1 gene had 4774bp with four introns (47, 52, 54 and 46bp). The N-terminal part of the protein displayed a conserved sequence controlling both sugar donor and accepter for substrate specificity whereas its C-terminal part carried a DXD motif that coordinated donor sugar binding. After complete removal of the gene UGT1, the mutant 1152-3 still produced 27.7±3.1g/L of pullulan and 4.6U/g of the specific glucosyltransferase activity while its wild type strain P16 yielded 63.38±2.0g/L of pullulan and 5.7U/g of the specific glucosyltransferase activity. However, after overexpression of the gene UGT1, the transformant G63 could produce 78.0±3.01g/L of pullulan and 19.0U/g of the specific glucosyltransferase activity. It is interesting to note that the molecular weight of the produced pullulan by the wild type strain was 4.6×105 while that of the produced pullulan by the transformant G63 was 6.2×105. During the 10-Litter fermentation, the pullulan titer produced by the transformant G63 reached 80.2±2.0 g/L within 132h.