Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 ℃, aeration rate 6 Lmin-1 and agitation speed 250 rmin-1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

Herbicidal activity of Aureobasidium pullulans PA-2 on weeds and optimization of its solid-state fermentation conditions

Fungal strain PA-2 was isolated from infected poplar leaves from the Ping’an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer(ITS) sequence of its 16S r DNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat(Triticum aestivum), faba bean(Vicia faba), and barley(Hordeum vulgare). By contrast, oilseed rape(Brassica napus) and pea(Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose(C4), nitrogen source of soybean flour(N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development ofA. pulluans PA-2 as an herbicide for bio-control of the weed.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Intraspecific Diversity of Aureobasidium pullulans Strains from Different Marine Environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureo-basidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.

The Synergism of Chemical Herbicides and Aureobasidium pullulans for Control Cleavers(Galium aparine L.) in Wheat

Aureobasidium pullulans, a biocontrol agent for the annual weed Galium aparine L. was evaluated in vitro for its compatibility with commercial formulation of five herbicides at 1X （recommended field rate）, 0.5X, 0.2X, 0.1X 0.067X, and 0.05X concentrations. Germination of A. pullulans with paraquat, 2, 4-D, quizalofop-p, and ctethodim treatment appeared reduced compared with germination of A. pullulans with fluroxypyr treatment at all concentrations. Stunted and shorter germ tubes in comparison with the control were observed with 2, 4-D, quizalofop-p, and clethodim at 0.2X. All concentration of paraquat, 2, 4-D, quizalofop-p, and clethodim except 0.05X, significantly decreased radial growth of A. pullulans compared with its growth on the untreated PDA medium. Field trials to further develop A. pullulans as bio- control agent for control G. aparine L. was conducted to test the effectiveness of this fungus in wheat plots for 2 years at the same location in Xining. Treatments included spore suspensions of A. pullulans alone, a mixture of both fungus and fluroxypyr in wheat. Biocontrol agent effectiveness was estimated at approximately 7 and 14 days after treatment, as disease incidence, percent weed control, and weed biomass reduction. Significant reduction in weed biomass occurred in combination treatments, and potential exists to tank mix A. pullulans with fluroxypyr. Leaf surface moisture and air temperatures following application may account for inconsistencies in field results between years. This fungal organisms show potential as bioherbicides for weeds in G. aparine L.

Studies on the optimal culture conditions of <i>Aureobasidium pullulans</i>to produce exopolysaccharides

In the current study, in order to change the permeability of cell membrane and solve the problem of linked group of fungi mycelium, the method of adjusting osmotic pressure of medium and adding tween-80 was established. The utilized strain with relatively high exopolysaccharide (EPS) yield and low pigment level was obtained after the rejuvenation and sifting of long-preserved Aureobasidium pullulans strain. The optimal proportion of substrate was determined by means of orthogonal test. The transformation ratio of EPS was increased by 10% - 20% and the pigment content was greatly reduced. The fermenting liquor is between creamy white and pale yellow, and the white primary product can be gained without decolourization step. Furthermore, to magnify to 5 L bioreactor can get the similar result.

The ornithine-urea cycle involves fumaric acid biosynthesis in Aureobasidium pullulans var.aubasidani,a green and eco-friendly process for fumaric acid production

The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming.In this study,the engineered strains of A.pullulans var.aubasidani were found to be suitable for green fumaric acid producer.Removal and complementation of the relevant genes showed only the ornithine-urea cycle(OUC)was involved in high level fumarate biosynthesis which was controlled by the Ca^(2+)signaling pathway.Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1±4.3 g/L of fumarate at flask level and 93.9±0.8 g/L of fumarate during the fed-batch fermentation.As a yeast-like fungal strain,it was very easy to cultivate A.pullulans var.aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production.It was found that 2 mol of CO_(2) could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC.Therefore,the OUC-mediated fumarate biosynthesis pathway in A.pullulans var.aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.

Improved viability of trehalose on Lactobacillus plantarum embedded with whey protein concentrate/pullulan in simulated gastrointestinal conditions and its application in acid juice

Trehalose(TRE)was used to improve the gastrointestinal tolerance of Lactobacillus plantarum embedded with whey protein concentrate/pullulan(WPC/PUL)hydrogel and the embedded L.plantarum was applied to juice.The study indicated that 5%TRE significantly increased the viable counts of L.plantarum embedded in WPC/PUL hydrogel from(8.83±0.03)to(9.14±0.04)(lg(CFU/g))in simulated gastric juice(SGJ)and from(9.13±0.04)to(9.38±0.04)(lg(CFU/g))in simulated intestinal juice,respectively.The addition of TRE improved the glass transition temperature of WPC/PUL hydrogel and decreased the hardness and its solubility in SGJ,which may be responsible for the improved protection of WPC/PUL hydrogels on L.plantarum.In addition,TRE increased the viable counts of L.plantarum in WPC/PUL probiotic microcapsule juice at low pH and high temperature during storage.

Screening, purification, and characterization of an extracellular lipase from Aureobasidium pullulans isolated from stuffed buns steamers

An extracellular lipase from Aureobasidium pullulans was obtained and purified with a specific activity of 17.7 U/mg of protein using ultrafiltration and a DEAE-Sepharose Fast Flow column. Characterization of the lipase indicated that it is a novel finding from the species A. pullulans. The molecular weight of the lipase was 39.5 kDa, determined by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE). The enzyme exhibited its optimum activity at 40 °C and pH of 7. It also showed a remarkable stability in some organic solutions(30%, v/v) including n-propanol, isopropanol, dimethyl sulfoxide(DMSO), and hexane. The catalytic activity of the lipase was enhanced by Ca2+ and was slightly inhibited by Mn2+ and Zn2+ at a concentration of 10 mmol/L. The lipase was activated by the anionic surfactant SDS and the non-ionic surfactants Tween 20, Tween 80, and Triton X-100, but it was drastically inhibited by the cationic surfactant cetyl trimethyl ammonium bromide(CTAB). Furthermore, the lipase was able to hydrolyze a wide variety of edible oils, such as peanut oil, corn oil, sunflower seed oil, sesame oil, and olive oil. Our study indicated that the lipase we obtained is a potential biocatalyst for industrial use.

Dual pH and microbial-sensitive galactosylated polymeric nanocargoes for multi-level targeting to combat ulcerative colitis

Ulcerative colitis(UC)is a type of inflammatory bowel disease characterized by inflammation,ulcers and irritation of the mucosal lining.Oral drug delivery in UC encounters challenges because of multifaceted barriers.Dexamethasone-loaded galactosylated-PLGA/Eudragit S100/pullulan nanocargoes(Dexa-GP/ES/Pu NCs)have been developed with a dual stimuli-sensitive coating responsive to both colonic pH and microbiota,and an underneath galactosylated-PLGA core(GP).The galactose ligand of the GP preferentially binds to the macrophage galactose type-lectin-C(MGL-2)surface receptor.Therefore,both stimuli and ligand-mediated targeting facilitate nanocargoes to deliver Dexa specifically to the colon with enhanced macrophage uptake.Modified emulsion method coupled with a solvent evaporation coating technique was employed to prepare Dexa-GP/ES/Pu NCs.The nanocargoes were tested using in vitro,ex vivo techniques and dextran sodium sulfate(DSS)induced UC model.Prepared nanocargoes had desired physicochemical properties,drug release,cell uptake and cellular viability.Investigations using a DSS-colitis model showed high localization and mitigation of colitis with downregulation of NF-ĸB and COX-2,and restoration of clinical,histopathological,biochemical indices,antioxidant balance,microbial alterations,FTIR spectra,and epithelial junctions’integrity.Thus,Dexa-GP/ES/Pu NCs found to be biocompatible nanocargoes capable of delivering drugs to the inflamed colon with unique targeting properties for prolonged duration.

Biocontrol Efficacy of Three Antagonistic Yeasts Against Penicillium expansum in Harvested Apple Fruits

Three antagonistic yeasts, Trichosporon pullulans (Lindner.) Diddens and Lodder, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) F. C. Harrison, selected from over 50 yeasts on apple fruits, were investigated of their biocontrol efficacy against blue mould rot caused by Penicillium expansum Link in apple fruits. T. pullulans was identified to be a new yeast antagonist and was the most effective at inhibiting blue mould rot in the three yeasts. The effects of different concentrations of the three yeasts and addition Of nutrients, as well as combination with calcium on controlling blue mould rot in apples were presented in the paper. The results indicated that the higher the yeast concentrations, the more effective the yeasts on biocontrol activity. Addition of nutrients reduced the biocontrol capacity of the yeasts. Combination of 0.18 mol/L CaCl2 with C. laurentii cell suspensions could significantly enhance its effect to P. expansum in apple fruits, while the efficacy of calcium on biocontrol activity of T. pullulans or R. glutinis was not remarkable.

Pullulan分子结构的研究

利用 GC- MS、IR和 13 C- NMR对 Pullulan的连接碳位和键型进行了研究 ,结果表明 ,Pullulan分子的主链由 β(1→ 3)糖苷链相连 ,主链上每 2～ 3个葡萄糖带有一个葡萄糖残基分枝 ,与主链以 β(1→ 6 )链相连 。

胞外多糖pullulan处理养猪场污水效果分析

用pullulan处理养猪场污水 ,结果表明 :污水的稀释倍数与pullulan的添加量对处理效果有极显著的影响。当Pullulan在 2倍稀释污水中的添加量达到 3mg/kg时 ,污水的生物需氧量 (BOD)、化学耗氧量 (COD)、总P、N的去除率达到 90 %以上 ;大肠杆菌的去除率达到 95 %以上 ,透过率也从 8%上升到 92 %。

胞外多糖Pullulan处理养猪场污水的研究

对 Pullulan处理养猪场污水进行了研究 .结果表明 :污水的稀释倍数与 Pullulan的添加量对处理效果有极显著的影响 ,当 Pullulan在 2倍稀释污水中的添加量达到 3mg/ kg时 ,污水的 BOD、COD、总 P、N的去除率达到 90 %以上 ;大肠杆菌的去除率达到 95 %以上 ,透过率也从 8%上升到 92 % .

动力学原理在芒果涂膜保鲜中的应用

通过对采后芒果果实经Pullulan涂膜处理后的芒果果实呼吸作用的变化的研究,利用芒果呼吸作用产生的CO2浓度(分压)建立芒果呼吸强度的动力学方程。研究结果表明:芒果经3%Pullulan溶液涂膜后的呼吸强度的动力学方程式为:RCO2=6.15×10-2/y2.2723;未经处理的呼吸强度的动力学方程式:RCO2=0.331/y1.9112。建立此方程式为探讨最佳的涂膜材料、涂膜厚度提供依据。

麦芽三糖的制备与分析

目的 :以普鲁兰多糖为底物 ,制备麦芽三糖。方法 :在 46℃ ,p H5 .6时以普鲁兰酶特异性水解普鲁兰多糖的 α- 1,6糖苷键 ,酶解反应 8小时 ,经分级沉淀、浓缩、干燥 ,得到麦芽三糖干粉。以进口试剂为对照 ,通过纸层析及高效液相色谱对产物做定性、定量分析。结果 :以本法制备麦芽三糖 ,产物收率为 6 3.1% ,纸层析定性为麦芽三糖 ,HPL C测定其纯度为 92 .3%。结论 :以本法制备麦芽三糖工艺简单 ,经 HPL

麦芽四糖生产工艺试验

麦芽四糖是一种新型麦芽低聚糖．试验中采用麦芽四糖淀粉酶和普鲁兰酶协同水解预处理过的淀粉，通过单因素试验和响应面曲线（ＲＳＡ）法确定了较好的酶解工艺条件．所得产品中麦芽四糖占总糖比例达８４％以上。

胞外多糖Pullulan分子的结构研究

本试验利用CC-MS、IR和^13C-NMR对Pullulan的连接碳位和键型进行了研究，结果表明，Pullulan分子的主链由β(1→3)糖苷链相连，主链上每2～3个葡萄糖带有一个葡萄糖残基分枝，与主链以β(1→6)链相连，Pullulan分子上还含有羧基。

Pullulan的抗肿瘤活性研究

本文对Ｐｕｌｌｕｌａｎ抗小鼠移植性Ｍｅｔｈｙｌｃｈｏｌａｎｔｈｒｅｎｅ－Ｉ Ｏｉｌｇｏｃｅｌｌｕｌｅｒ和Ｓｏｌｉｄ Ｓａｒｃｏｍａ－１８０肿瘤的活性进行了研究，结果表明，Ｐｕｌｌｕｌａｎ的给与剂量和给与方式不同，其抗肿瘤效果也不同，从抗Ｍｅｔｈｙｌｃｈｏｌａｎｔｈｒｅｎｅ－Ｉ Ｏｉｌｇｏｃｅｌｌｕｌｅｒ来说，腹腔给与的抗肿瘤效果显著高于口投给与之效果，当腹腔给与的剂量为０．

5-Fluorouracil-loaded Self-assembled pH-sensitive Nanoparticles as Novel Drug Carrier for Treatment of Malignant Tumors

In order to improve the cancer-targeting and selective activity of antineoplastic agent [5-fluorouracil （5-FU）], a novel pH-responsive drug delivery system [pullulan acetate/sulfonamide （PA/SDM） conjugate] was synthesized by a diafiltration method. Sulfonamide was grafted to the hydrophobicaUy modified pullulan acetate to enhance the pH sensitivity for better cancer-targeting delivery. 5-FU was loaded into the self-assembled nanoparticles by the same method. The drug-loaded self-assembled nanoparticles were successfully obtained and characterized in terms of particle size, morphology and drug loading and release profile at various pHs. The results showed that the mean diameter of the self-assembled particles was approximately 100nm, with uniform size and good spherical morphology. The nanoparticles showed good stability at pH 7.4, which is equal to that of the normal body fluid, but shrank and aggregated below pH 6.8, which is close to the pH with tumors. The loading efficiency and concentration of released 5-FU was monitored at 269 nm on the UVNis spectrophotometer. The release profile was heavily pH-dependent around phvsiological pH, and the release rate was significantly enhanced under pH of 6.8.