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.

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.

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.

短梗霉(Aureobasidium pullulans)与短梗霉多糖发酵

短梗霉多糖系短梗霉发酵产物,它具有巨大的经济价值。短梗霉是一种多形态真菌,具有复杂的生活史,并且其形态学变化与其多糖合成之间关系密切。本文就短梗霉及其发酵特点进行较详细的综述。

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.

出芽短梗霉(Aureobasidium pullulans)生物合成普鲁兰多糖的研究进展

普鲁兰多糖是由出芽短梗霉(Aureobasidium pullulans)发酵产生的一种胞外多糖,其特殊的结构赋予了它许多优良的理化性质,广泛应用于食品、制药、化工、石油及其他制造业。在普鲁兰多糖生物合成过程中,普鲁兰多糖的化学结构、产率及颜色的深浅等受到多种营养条件和环境因素的影响。介绍了普鲁兰多糖的结构、性质,重点综述了国内外学者对发酵条件优化的研究现状,最后总结了普鲁兰多糖生物合成研究的发展方向。

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.

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.

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.

Aureobasidium pullulans UVMU3-1产耐热性胞外漆酶活性物质培养基优化及其对染料脱色作用

对出芽短梗霉无色素突变株Aureobasidium pullulans UVMU3-1产耐热性胞外漆酶活性物质培养基进行优化,并研究其分子量大小、热稳定性及对三苯甲烷类染料孔雀石绿和结晶紫的脱色作用.结果表明:蔗糖、NaNO_3为UVMU3-1产耐热性胞外漆酶活性物质的最佳碳源和氮源,产酶优化培养基配方为:蔗糖66.5 g·L^(-1),NaNO_338.4 g·L^(-1),NaCl 1.08 g·L^(-1),酵母浸粉0.2 g·L^(-1),KH_2PO_41 g·L^(-1),pH=6.0.影响UVMU3-1产耐热性胞外漆酶活性物质的因素依次为:起始pH>蔗糖>NaCl>NaNO_3,培养7 d时产生最大酶活(94.81 U·L^(-1)),比对照提高3.85倍.其分子量小于1000 D,具有良好的热稳定性,煮沸30 min后高温酶活仍残留72.50%.粗酶液(7.5 U·L^(-1))与三苯甲烷类代表性染料孔雀石绿和结晶紫75℃反应5 min后,脱色率分别为76.3%、77.5%,反应35 min后脱色率分别达到96.3%、95.3%.

A New Pullulan-Producing Yeast and Medium Optimization for Its Exopolysaccharide Production

Yeast strain Y68 producing high level of pullulan was isolated from the phyton collected in Toulouse, France. This strain was identified to be Rhodotorula bacarum by BIOLOG analysis. This is the first report that pullulan was produced by Rhodotorula bacarum. The optimal medium (g L -1) for pullulan production by this strain was 80 glucose, 20 soybean cake hydrolysate, 5 K 2HPO 4, 1 NaCl, 0.2 MgSO 4·7H 2O, 0.6 (NH 4) 2SO 4, pH 7.0. Under this condition, 54 g L -1 pullulan was produced within 60 h at 30 ℃. Pullulan is a better starting material for producing marine prodrugs.

Enhanced primary treatment of low-concentration municipal wastewater by means of bio-flocculant Pullulan

Jar tests were conducted to investigate the performance of enhanced primary treatment processes for low-concentra-tion municipal wastewater from South China by using composite flocculant combined with bio-flocculants Pullulan and poly-aluminum-chloride (PAC). The optimum dosage for composite flocculant and conditions for flocculation were determined. The experimental results indicated that composite flocculant had high efficiency for removing over 95% of turbidity, over 58% of CODCr (chemical oxygen demand determined with potassium dichromate), over 91% of TP (total phosphate), and over 15% of NH3-N. Moreover, it could improve sludge settling and dehydration properties, and decrease the treatment cost.

Electrospun Food-Grade Ultrafine Fibers from Pectin and Pullulan Blends

Electrospinning was used to produce ultrafine fibers and fibrous mats from aqueous solutions containing two edible polysaccharides: pectin (PEC) and pullulan (PUL). The process excluded the use of a synthetic carrier polymer or non-aqueous solvents thus maintaining the food-grade status of the components. The inclusion of PUL reduced the surface tension and electric conductivity of pectin solution, and promoted molecular entanglement between PEC and PUL as confirmed by rheological analysis. The spinnability of either polysaccharide was promoted by inclusion of the other. Cross-linking PEC networks were obtained by soaking the fibrous mats in successive Ca2+ solution. The fibrous mats can be used to carry bioactive compounds as demonstrated using probiotic bacteria Lactobacillus rhamnosus GG (LGG) as a model bioactive compound. The research is significant for the development of food products with unique textures and functionalities.

Effect of Pullulan and Hydrocolloids on Rheological Properties and Quality Parameters of Wheat-Soy Baladi Bread

Baladi bread is a staple food in Egypt and high nutritive value however, stales rapidly. The aim of the study was to conduct a comparative evaluation of pullulan and hydrocolloid combinations on rheological properties and quality parameters of wheat-soy baladi bread. Pullulan polysaccharides (Pu) and hydrocolloids as Arabic gum (AG), K-carrageenan (KC), pectin (P), and xanthan gum (XG) were applied. Rheological properties of samples included Pu and hydrocolloids were analyzed through extensiograph and farinograph. The crust color, staling, and sensory attributes of bread were evaluated. Results revealed that wheat flour (WF) has lower water absorption capacity, dough development time, mixing tolerance index, and resistance to extension however, has higher dough stability, extensibility, and dough energy compared to wheat-soy flour (WSF). Pu and/or hydrocolloids addition to WSF dough evidently improved bread quality. Pu was significantly enhanced crust color and yielded fresher bread relative to control. Additionally, Pu showed the highest Alkaline Water Retention Capacity (AWRC) value after 5 days at 25°C ± 2°C. Results demonstrated that hydrocolloids especially pullulan can significantly improve dough properties, baking quality, sensory acceptability, and delay staling of wheat-soy baladi bread.

负载互叶白千层精油的Zein/Pullulan复合膜乳液对芒果保鲜贮藏品质的影响

为研究新型复合乳液对芒果贮藏品质的影响,制备了负载0、1%、2%(v/v)互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖复合乳液,并对芒果进行涂膜处理,于20℃、RH 65%贮藏,定期测定指标。结果表明:3种复合乳液涂膜处理均能减缓芒果失重率的升高,且维持更高的果肉Vc含量,但对果肉TA和TSS含量影响不大;而负载互叶白千层精油的复合乳液还能有效地抑制芒果果实的呼吸作用,延缓果皮叶绿素的降解和类胡萝卜素含量的升高,其中负载1%互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖乳液处理在延缓果实叶绿素的降解、减缓失重率的上升和维持Vc含量方面的效果更好。综上,负载1%互叶白千层精油的玉米醇溶蛋白/普鲁兰多糖乳液更利于维持芒果的贮藏品质。

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.

共表达分子伴侣蛋白提高III型普鲁兰水解酶在短小芽孢杆菌中的分泌表达及发酵优化

本研究旨在通过共表达分子伴侣蛋白以及优化发酵条件来提高III型普鲁兰水解酶(TK-PUL)在短小芽孢杆菌中的分泌表达水平。通过构建共表达TK-PUL和分子伴侣蛋白的多种重组短小芽孢杆菌,并以胞外酶活为指标对其进行筛选,确定最有利于TK-PUL分泌表达的分子伴侣蛋白及对应的重组短小芽孢杆菌。在此基础上采用单因素实验和响应面法优化重组短小芽孢杆菌的发酵条件。结果表明,共表达分子伴侣蛋白PrsA^(Ba)的重组短小芽孢杆菌的胞外酶活达到98.79 U/mL,提高了0.31倍。该重组短小芽孢杆菌的最佳培养基包括19.65 g/L葡萄糖、21.46 g/L酵母提取物、12.01 g/L MgCl_(2)·6H_(2)O、9.02 g/L脯氨酸、0.01 g/L FeSO4·7H_(2)O、0.01 g/L MnSO4·4H_(2)O、0.001 g/L ZnSO4·7H_(2)O。在发酵温度为35℃、起始发酵pH为7.0的条件下,该重组短小芽孢杆菌于最佳培养基中培养66 h时,其胞外酶活达到192.68 U/mL,提高了1.56倍。本研究通过共表达分子伴侣蛋白和发酵优化实现了TK-PUL在短小芽孢杆菌的高效分泌表达,为TK-PUL的应用提供了基础。

出芽短梗霉LHS-m022黑色素葡聚糖的发酵影响因素和生物活性

为探索出芽短梗霉LHS-m022黑色素多糖的发酵影响因素和生物活性,采用菌种发酵、分离提取及生物检测方法,对发酵液性质、产物收率、生物转化率及生物活性进行测定分析。结果表明,LHS-m022黑色素多糖发酵的最佳碳、氮源分别是蔗糖和胰酪蛋白胨,蔗糖最佳浓度为60 g/L;发酵培养基中诱导剂L-多巴的最适添加量为2.0 g/L,发酵液黑变活性和生物转化率分别是2.481和71.93%;细胞通透剂鼠李糖脂的最适添加量是0.021μL/L,发酵液黑变活性和生物转化率分别高达2.794和73.9%。全波长扫描、FTIR与HPLC分析表明,WAI为黑色素,PsB为黑色素葡聚糖结构。研究结果揭示,粗黑色素葡聚糖样品经121℃以上高温处理,仍然得到95.37%的絮凝率。采用1.50和2.00 g/L的样品浓度进行检测,分别得到99.55%的羟自由基清除活性和99.00%的抗氧化活性。

一株高产普鲁兰多糖短梗霉菌株的基因组分析及产糖机理探究

从分子水平解析微生物的基因组变化,从而对其进行遗传改造以提高普鲁兰多糖的产量是目前亟待解决的问题。通过比较基因组学方法,对一株高产普鲁兰多糖的出芽短梗霉(TN-31)与其他短梗霉菌株进行了基因组比较。结果显示,该菌株发生了基因组加倍,其基因组中存在大量的遗传变异,这可能是其高产普鲁兰多糖的重要原因。此外,该菌株的基因组大小高于其他短梗霉菌株,其特有基因和功能基因启动子区的特殊调控序列可能赋予其不同于其他短梗霉菌株的特性。这种特殊表型可能是适应性进化的结果,通过遗传变异和适应性选择,能够适应其所处的环境。