普鲁兰多糖应用现状研究

介绍了普鲁兰多糖的理化性质和生理功能,阐述了其在农产品、食品、环保、包装、医药、石油等领域的应用现状,并对其良好的市场前景进行了展望。     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.     

普鲁兰酶的研究进展

普鲁兰酶是一类淀粉脱支酶,能够专一性切开支链淀粉分支中的α-1,6糖苷键,形成直链淀粉。普鲁兰酶可与其他淀粉酶协同作用,在淀粉加工工业中有着重要的用途和良好的市场前景。本文就普鲁兰酶的结构与催化机理、酶学性质、来源和应用进行了综述。     

普鲁兰酶改性瓜尔胶及其与黄原胶复配性能研究

通过红外光谱表征、稳态流动和动态应力扫描法考察了普鲁兰酶对瓜尔胶的脱支效应和改性瓜尔胶与黄原胶的复配性能。结果表明,普鲁兰酶不仅降低了瓜尔胶中半乳糖的含量,也降低了瓜尔胶的分子量及粘度,但未改变瓜尔胶主链的主体结构和糖苷键的构型,也未改变其流变学性质。与天然瓜尔胶-黄原胶复配胶相比,改性瓜尔胶-黄原胶复配胶表现出了较强的屈服应力,能在更高的压力范围内维持较高的弹性模量。随着混合温度升高,复配胶的弹性模量逐渐增大,并且在60℃时达到最大值。研究说明,普鲁兰酶改性瓜尔胶与黄原胶复配具有显著的协同增效效应。     

普鲁兰菌发酵条件的研究

本文对普鲁兰菌的发酵条件进行了研究,并总结出其高产普鲁兰多糖的条件。实验结果,普鲁兰多糖产率由６％,提高并稳定在７０％左右,最高达７６％。本文对影响普鲁兰多糖高产的发酵条件进行了分析。     

普鲁兰酶的异源表达、结构解析及分子改造研究进展

淀粉是由葡萄糖单元通过α-1,4-葡萄糖苷键和α-1,6-葡萄糖苷键连接而成,不仅是食物的主要成分,也是淀粉深加工工业的基本原料来源。普鲁兰酶能够高效水解淀粉分子中的α-1,6-葡萄糖苷键,与其他的淀粉加工酶复合使用,能够有效提高淀粉的利用率,在淀粉深加工工业中具有“提质增效”的重要作用。本文综述了普鲁兰酶产酶菌株的筛选及编码基因的克隆表达,总结了表达元件及发酵条件优化对普鲁兰酶产酶水平的影响,探讨了普鲁兰酶结构解析及分子改造等方面的研究进展。同时分析了当前研究中存在的问题,并对未来的研究进行了展望,以期为普鲁兰酶的研究及应用提供参考和启示。     

微生物GH13家族淀粉脱支酶研究进展

普鲁兰酶和异淀粉酶都具有典型的(β/α)8桶状结构,属于GH13家族淀粉脱支酶.GH13家族的淀粉脱支酶能够专一、高效地水解淀粉分支部位的α-1,6-糖苷键,可以有效提高淀粉原料利用率和生产效率,在淀粉加工工业中具有重要的应用价值,因此近年来对GH13家族淀粉脱支酶的研究逐渐增多.本文系统地综述了微生物来源的GH13家族淀粉脱支酶的国内外研究进展,分别对普鲁兰酶和异淀粉酶的底物特异性及结构基础、研究现状以及应用和研究新趋势进行了阐述.并对GH13家族淀粉脱支酶研究中存在的问题和下一步开发方向提出了见解.     

产酸克雷伯氏菌M5al普鲁兰酶的异源表达及酶学性质研究

普鲁兰酶(EC 3.2.1.41)是一类淀粉脱支酶,能够特异性水解淀粉中的α-1,6-糖苷键,从而提高淀粉的利用率,在以淀粉为原料的食品、纺织、生物燃料和洗涤剂等行业中具有重要的应用价值。本研究以产酸克雷伯氏菌Klebsiella oxytoca M5al基因组DNA为模板,将PCR扩增得到的普鲁兰酶基因pul A克隆至表达载体p ET28a(+),构建好的重组质粒转化大肠杆菌Escherichia coli BL21(DE3),在培养基中添加0.5 mmol/L异丙基硫代半乳糖苷(IPTG)的条件下对该酶基因进行诱导表达,经镍柱纯化获得重组普鲁兰酶用于酶学性质研究。SDS-PAGE及Western Blot检测显示普鲁兰酶基因pul A在上述大肠杆菌宿主中成功获得了表达。该重组酶最适反应p H5.5,最适温度60℃。金属离子对酶活性有一定影响。Mn2+对酶活促进作用显著;Fe3+、Mg2+、Fe2+对酶活只有微弱的促进作用,而Cu2+对酶活造成强烈抑制。来源于Klebsiella oxytoca M5al的普鲁兰酶最适催化条件符合工业生产中淀粉糖化工艺的要求,具有应用于淀粉工业的潜力。     

生物合成材料聚β-羟基丁酸(PHB)的研究进展

聚β-羟基丁酸(PHB)是原核微生物在碳、氮营养失衡的情况下,作为碳源和能源贮存在生物体内的一类热塑性聚酯.它作为微生物合成的可降解材料,除了具有与化学合成高分子相似的性质外,还具有一般化学合成高分子没有的性质,如光学活性好、透氧性低、抗紫外线辐射、生物可降解性、生物组织相容性、压电性和抗凝血性等,具有广阔的应用前景,越来越受到人们的关注.国内外的许多公司和科研机构纷纷开展可降解塑料的研发工作.着重介绍了PHB的理化性质、检测方法、生物合成、降解以及基因改良菌种方面的研究进展,同时对其应用、目前存在的问题以及可能的解决方案进行了讨论.     

长野芽孢杆菌普鲁兰酶的同源建模及三维结构分析

普鲁兰酶(Pullulanase)是脱支酶,因其能水解葡聚糖的α-1,6-糖苷键而有不同的工业应用潜力。本研究通过同源建模和分子对接的方法对长野芽孢杆菌(Bacillus naganoensis)普鲁兰酶进行建模及其三维结构分析,表明该酶由CBM41-X45aX25-X45b-CBM48-GH13_14多结构域组成,酶蛋白中心形成其催化区,催化区的Asp619、Glu648和Asp733三个残基构成酶的催化三联体。同时,通过柔性对接研究了酶与底物分子相互作用的关系,并预测构成酶的活性中心相关氨基酸残基,为进一步改良酶的特性提供重要的理论依据。     

Effects of plastic composite support and pH profiles on pullulan production in a biofilm reactor

Pullulan is a linear homopolysaccharide which is composed of glucose units and often described as α-1, 6-linked maltotriose. The applications of pullulan range from usage as blood plasma substitutes to environmental pollution control agents. In this study, a biofilm reactor with plastic composite support (PCS) was evaluated for pullulan production using Aureobasidium pullulans. In test tube fermentations, PCS with soybean hulls, defatted soy bean flour, yeast extract, dried bovine red blood cells, and mineral salts was selected for biofilm reactor fermentation (due to its high nitrogen content, moderate nitrogen leaching rate, and high biomass attachment). Three pH profiles were later applied to evaluate their effects on pullulan production in a PCS biofilm reactor. The results demonstrated that when a constant pH at 5.0 was applied, the time course of pullulan production was advanced and the concentration of pullulan reached 32.9 g/L after 7-day cultivation, which is 1.8-fold higher than its respective suspension culture. The quality analysis demonstrated that the purity of produced pullulan was 95.8% and its viscosity was 2.4 centipoise. Fourier transform infrared spectroscopy spectra also supported the supposition that the produced exopolysaccharide was mostly pullulan. Overall, this study demonstrated that a biofilm reactor can be successfully implemented to enhance pullulan production and maintain its high purity.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.     

Enhanced pullulan production in a biofilm reactor by using response surface methodology

Pullulan is a linear homopolysaccharide that is composed of glucose units and often described as α-1, 6-linked maltotriose. In this study, response surface methodology using Box–Behnken design was employed to study the effects of sucrose and nitrogen concentrations on pullulan production. A total of 15 experimental runs were carried out in a plastic composite support biofilm reactor. Three-dimensional response surface was generated to evaluate the effects of the factors and to obtain the optimum condition of each factor for maximum pullulan production. After 7-day fermentation with optimum condition, the pullulan production reached 60.7 g/l, which was 1.8 times higher than the result from initial medium, and was the highest yield reported to date. The quality analysis demonstrated that the purity of produced pullulan was 95.2%, and its viscosity was 2.5 centipoise (cP), which is higher than the commercial pullulan and related to its molecular weight. Fourier transform infrared spectroscopy (FTIR) also suggested that the produced exopolysaccharide was pullulan.     

Pullulan: biosynthesis,production, and applications

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.     

Production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82 with pH control and DL-dithiothreitol addition

Xylose, the second most abundant sugar in lignocellulosic materials, is not efficiently utilized in current lignocellulose biotransformation processes, such as cellulosic ethanol production. The bioconversion of xylose to value-added products, such as pullulan, is an alternative strategy for efficient lignocellulose biotransformation. This paper reports the production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82. The effects of DL-dithiothreitol (DTT) and pH on pullulan production from xylose were also intensively investigated. A maximal increase of 17.55% of pullulan production was observed in flasks added with 1.0 mM DTT. Batch fermentations with controlled pH were also conducted, and the optimal pH for cell growth and pullulan synthesis was 3.0 and 5.0, respectively. Based on these findings, two-stage pH control fermentations were performed, in which the pH of the medium was first adjusted to 3.0 for cell growth, and then changed to 5.0 for pullulan synthesis. However, the earlier the pH was changed to 5.0, the more pullulan was produced. Fermentation with controlled pH of 5.0 acquired the highest pullulan production. Under the optimized conditions (with the addition of 1.0 mM DTT and controlled pH of 5.0), the maximal pullulan production obtained from xylose was 17.63 g/L. A. pullulans AY82 also readily fermented hemicellulose hydrolysate under these optimized conditions, but with lower pullulan production (12.65 g/L). Fourier transform infrared spectroscopy and high-performance liquid chromatography showed that the structure of the pullulan obtained in this study was identical to that of the pullulan standard.     

Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases. Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).     

茁霉多糖发酵及提取工艺条件研究

目的：茁霉多糖是一种新型多功能生物材料,该文拟对出芽短梗霉发酵茁霉多糖及其提取条件进行初步探索。方法：通过摇瓶培养确定了该菌株的发酵优化条件,在此条件下,获得了较高的多糖产量,同时通过醇提的方法对茁霉多糖的提取进也行了研究。结果：初步确定了茁霉多糖最佳发酵与提取条件。结论：摇瓶转速和发酵初始pH值是多糖发酵的重要影响因素,它们与多糖的合成密切相关。     

Synthesis of Pullulan by Acetone-dried Cells and Cell-free Enzyme from Pullularia pullulans,and the Participation of Lipid Intermediate

It was demonstrated that the polysaccharide, pullulan, was synthesized from sucrose by acetone-dried cells of Pullularia pullulans or from UDPG by cell-free enzyme preparations prepared from the organism. The pullulan formed was estimated by precipitation with ethanol, and determining maltotriose produced after treating the precipitate with Aerobacter isoamylase (pullulanase). Acetone cells (5 g) shaken with 200 ml of 10% sucrose produced over 250 mg of pullulan per 100 ml after 90 hr at 30°C and pH 6.0. Cell-free enzyme produced pullulan from UDPG in the presence of ATP. ATP was essential for the biosynthesis, and ADPG could not replace for UDPG.

In addition, it was observed that a lipid containing glucose residue was formed during, the reaction. The nature of this glucolipid was examined, and possible participation of a lipid intermediate was assumed in the pullulan biosynthesis.     

High pullulan yield is related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68

Effects of different pH and carbon sources on pullulan production, UDP-glucose level and pullulan-related synthases activity inAureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium with initial pH 7.0 than when it was grown in the same medium with constant pH 6.0. The results also show that higher pullulan yield was obtained when the cells were grown in the medium containing glucose than when they were cultivated in the medium supplementing other carbon sources. Our results demonstrate that the more pullulan was synthesized, the less UDP-glucose was left in the cells ofA. pullulans Y68. However, it was observed that more pullulan was synthesized; the cells had higher pullulan-related synthase activity. Therefore, high pullulan yield was related to low UDP-glucose level and high pullulan-related synthases activity inAureobasidium pullulans Y68.     

Estimation of pullulan by hydrolysis with pullulanase

A novel method for the estimation of pullulan was developed in which pullulan was hydrolysed by pullulanase. The hydrolysed product was mainly maltotriose and was determined colorimetrically using 3,5-dimethylsalicylic acid. This gave good linearity with respect to the concentration of pullulan in the fermentation broth. The content of pullulan determined in this way was less than that determined by a coupled enzyme assay and was comparable to that determined by an HPLC method. The new method was specific for estimation of pullulan, demonstrated high accuracy, and could assay pullulan from up to 3.2 mg/ml.