Pullulan Production Research Articles

The Biocatalytic Process of Pullulan Production by Auerobasidium pullulans Cells Being in a Form of High Density Populations

The results of investigation and comparative analysis of biocatalytic process of pullulan production by a high cell density populations of Auerobasidium pullulans cells taken in suspended and immobilized into poly(vinyl alcohol) cryogel forms are performed in the work. The possible effective using of the immobilized biocatalyst was demonstrated and the characteristics of the process of pullulan production from different hydrolisates of renewable raw materials such as tubers of Jerusalem artichoke, aspen, Chlorella vulgaris biomass, potato pulp were determined. It was revealed that A. pullulans cells entrapped into poly(vinyl alcohol) cryogel can 1.5-times faster convert glucose to pullulan and accumulate the polysaccharide in cultural broth in 1.7-times higher concentration as compared to suspended cells. Immobilized cells appeared to retain a high functional metabolic action during at least 15 working cycles with minimum (not more than 10 %) loss of activity. The analysis of experimental data confirmed that use of cells immobilized into poly(vinyl alcohol) cryogel enables decrease in 1.5–3 times the duration of the working cycle in the same process while achieving comparable level of product yield.

Simultaneously enhanced production of poly(β‐malic acid) and pullulan using a dissolved oxygen shift (DO‐shift) control strategy

AbstractBACKGROUNDPoly(β‐malic acid) (PMA) and pullulan are two valuable biopolymers that can be produced simultaneously by certain strains of pullulans. In previous studies, PMA production was successfully improved with reducing pullulan production. In the present study, a DO‐shift control strategy was developed to obtain simultaneous high yields of PMA and pullulan.RESULTSPMA and pullulan co‐production at various constant DO levels ranging from 10% to 90% saturation were carried out in a 5 L stirred tank fermentor. Based on the analysis of batch fermentation kinetics at various constant DO levels, a DO‐shift control strategy that shifted the DO level from 30% saturation to 70% saturation at 72 h was developed to effectively obtain high productions of both PMA and pullulan.CONCLUSIONBy applying the DO‐shift control strategy, the maximum concentrations of PMA and pullulan reached 118.6 g L−1 and 27.2 g L−1, respectively. PMA production was successfully improved without sacrificing pullulan production. © 2016 Society of Chemical Industry

A glycosyltransferase gene responsible for pullulan biosynthesis in Aureobasidium melanogenum P16

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.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose.

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

CreA is directly involved in pullulan biosynthesis and regulation of Aureobasidium melanogenum P16.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93±1.33g/l pullulan from 120g/l of glucose, while its wild-type strain P16 produced only 52.0±1.95g/l pullulan within 132h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.

Exploitation of Agricultural Wastes and By-Products for Production of Aureobasidium pullulans Y-2311-1 Xylanase: Screening, Bioprocess Optimization and Scale Up

The potential of several agricultural wastes and by-products (wheat bran, oat bran, corn cob, brewer’s spent grain, malt sprout, artichoke stem, sugar beet pulp, olive seed, cotton stalk and hazelnut skin) was examined as the substrate for xylanase production by Aureobasidium pullulans Y-2311-1. Based on the screening studies, wheat bran was selected as the best substrate for further optimization studies. The effects of initial medium pH, temperature and incubation time on xylanase production in shake flask system were optimized by response surface methodology (RSM). The optimum levels of the process variables defined by the model (initial medium pH, 4.24; temperature, 30.27 °C; and incubation time 126.67 h) resulted in production of 85.19 U/ml xylanase. Taking the RSM optimized parameters in shake-flask scale into consideration; xylanase production was scaled up to bioreactor system with a working volume of 1.5 l. The peak of enzyme production was achieved after 126 h incubation that has previously been determined by RSM studies at shake flask level. Furthermore, the optimum levels of agitation and aeration in bioreactor system was found as 200 rpm and 1.5 vvm. Maximum enzyme production was close to 85 kU/l which could be translated into a productivity of 0.68 kU/l/h. No previous work considered the statistical optimization of xylanase production by A. pullulans on wheat bran and scale up of the bioprocess to a bioreactor system.

Improving Erythritol Production of Aureobasidium pullulans from Xylose by Mutagenesis and Medium Optimization.

The biotransformation of xylose, the second most abundant sugar, has been a hot topic in recent years. In this work, Aureobasidium pullulans CGMCC3.0837 was subjected to UV mutagenesis to improve its erythritol production from xylose. The erythritol production of the obtained mutant ER35 was 50.92% (17.28g/L) higher than that of the parent strain. Response surface methodology was applied to optimize the medium composition. Yeast extract, KH2PO4, and citric acid were the key factors influencing erythritol synthesis, and the optimal concentrations were 17.82, 0.76, and 6.60g/L, respectively. Under the optimized conditions, 31.75 and 31.42g/L erythritol were obtained in shake flasks and in a 5-L fermentor, respectively. ER35 also showed a good consuming ability on xylose mother liquor with a final erythritol production of 26.35g/L. This report provided insights into the potential of A. pullulans for the production of erythritol using xylose as a carbon source.

Why sucrose is the most suitable substrate for pullulan fermentation by Aureobasidium pullulans CGMCC1234?

This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69g/L) also accumulated during early stages of fermentation (24h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. β-Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of β-fructofuranosidase production. However, β-fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells.

Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst

The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase.

The objective was to phylogenetically classify diverse strains of Aureobasidium pullulans and determine their production of feruloyl esterase. Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-β-1,4-xylanase overproduction were associated with phylogenetic clade 10 and particularly clade 8. Literature strains used for pullulan production all belonged to clade 7. These strains and 36 previously classified strains were tested for feruloyl esterase production, which was found to be associated with phylogenetic clades 4, 11, and particularly clade 8. Clade 8 strains NRRL 58552 and NRRL 62041 produced the highest levels of feruloyl esterase among strains tested. Production of both xylanase and feruloyl esterase are associated with A. pullulans strains in phylogenetic clade 8, which is thus a promising source of enzymes with potential biotechnological applications.

Characterization of UGPase from Aureobasidium pullulans NRRL Y-12974 and Application in Enhanced Pullulan Production.

UDPG pyrophosphatase (UGPase) plays an important role in carbohydrate metabolism, catalyzing a reversible production of uridine diphosphate glucose (UDPG) and pyrophosphate (PPi) from Glc-1-P and UTP. UGPase gene from Aureobasidium pullulans NRRL Y-12974 was cloned, overexpressed in Escherichia coli. The recombinant UGPase possess molecular mass of 55 KDa and specific activity of 7.33 U/mg protein. The K m values of rUGPase were 5.045 μM against UTP and 3.333 μM against Glc-1-P. The V max values of rUGPase were 3.467 μM min(-1)against UTP and 2.817 μM min(-1) against Glc-1-P. And, it does not catalyze Glc-1-P and ATP, nor galactose-1-P and UTP. Homolgous expression of UGPase in native organism can improve the intracellular UDPG concentration by 4.7-fold time. The yield of pullulan in engineering strain A4 was improved to 18.2 g g(-1) cell dry weight which is 1.3-fold time of parent strain. No obvious change of growth was found between engineering strain and parent strain. To the best of our knowledge, this is the first report of improving pullulan yield in A. pullulans using metabolic engineering technique.

Understanding the influence of Tween 80 on pullulan fermentation by Aureobasidium pullulans CGMCC1234

In this paper, several new perspectives concerned with the effect of Tween 80 promoting pullulan production were presented. With the presence of Tween 80, the maximum pullulan yield increased by 41% (53.04g/L). Meanwhile, the carbon source was consumed faster and the broth viscosity was higher. The lower final pH suggested that Tween 80 could protect the integrity of the mycelia. The dispersed filaments were not easily entangled with each other and less pellets were formed in the Tween 80 culture broth. FT-IR spectrum analysis indicated that the evaluated sample structure was coincided with commercial pullulan. The molecular weight of sample significantly dropped comparing with the control. The above findings indicated that Tween 80 facilitated the uptake of nutrient from surroundings to the microorganism and the release of pullulan into the extracellular fluid. These results were useful in better understanding the regulation and optimization of efficient pullulan fermentation.

Production of pullulan from raw potato starch hydrolysates by a new strain of Auerobasidium pullulans

In the present study, hydrolysis of potato starch with marine cold-adapted α-amylase and pullulan production from the hydrolysates by a new strain of Auerobasidium pullulans isolated from sea mud were conducted. The hydrolysis conditions were optimized as follows: reaction time 2h, pH 6.5, temperature 20°C, and α-amylase amount 12U/g. Under these optimum hydrolysis conditions, the DE value of the potato starch hydrolysates reached to 49.56. The potato starch hydrolysates consist of glucose, maltose, isomaltose, maltotriose, and trace of other maltooligosaccharides with degree of polymerization ranged 4–7. The maximum production of pullulan at 96h from the hydrolysate of potato starch was 36.17g/L, which was higher than those obtained from glucose (22.07g/L, p<0.05) and sucrose (31.42g/L, p<0.05). Analysis of the high performance liquid chromatography of the hydrolysates of the pullulan product with pullulanase indicated that the main composition is maltotriose, thus confirming the pullulan structure of this pullulan product.

Statistical modeling of pullulan production and its application in pullulan acetate nanoparticles synthesis

This work focused on the optimization of exo-polysaccharide, pullulan production by exploiting cassava bagasse, an agricultural solid waste residue by solid state fermentation and its application in the preparation of pullulan acetate nanoparticles. Statistical approach was investigated to maximize the pullulan production using C/N ratio, initial pH, NaCl and ZnSO4·5H2O. The optimum conditions for maximum yield of pullulan (39.42±0.62mg/gds) were found to be: C/N=25.94, initial pH=5.5 and NaCl=0.55g/L. Using the optimized medium variables, the production of pullulan was investigated in lab scale solid state fermentation. The pullulan produced was characterized by thermo gravimetric and XRD analysis. Also pullulan acetate nanoparticles were synthesized from chemical modification of pullulan and the average particle size of nanoparticles was examined by zeta particle sizer.

Central metabolic pathways of Aureobasidium pullulans CGMCC1234 for pullulan production

With the purpose of understanding the metabolic network of Aureobasidium pullulans, the central metabolic pathways were confirmed by the activities of the key enzymes involved in different pathways. The effect of different iodoacetic acid concentrations on pullulan fermentation was also investigated in this paper. The activities of phosphofructokinases and glucose-6-phosphate dehydrogenase existed in A. pullulans CGMCC1234, whereas 2-keto-3-deoxy-6-phosphogluconate aldolase activity was not detected. We proposed that the central metabolic pathways of A. pullulans CGMCC1234 included EMP and PPP, but no ED. Pullulan production declined fast as the iodoacetic acid increased, while cell growth offered upgrade firstly than descending latter tendency. Compared to the control group, the ratio of ATP/ADP of 0.60mM iodoacetic acid group was lower at different stages of pullulan fermentation. The findings revealed that low concentration of iodoacetic acid might impel carbon flux flow toward the PPP, but reduce the flux of the EMP.

Statistical optimization of process parameters for exopolysaccharide production by Aureobasidium pullulans using sweet potato based medium.

Statistical experimental designs were applied to optimize the fermentation medium for exopolysaccharide (EPS) production. Plackett–Burman design was applied to identify the significance of seven medium variables, in which sweet potato and yeast extract were found to be the significant variables for EPS production. Central composite design was applied to evaluate the optimum condition of the selected variables. Maximum EPS production of 9.3 g/L was obtained with the predicted optimal level of sweet potato 10 %, yeast extract 0.75 %, 5.5 pH, and time 100 h. The determined (R2) value was 0.97, indicating a good fitted model for EPS production. Results of this study showed that sweet potato can be utilized as a low-cost effective substrate for pullulan production in submerged fermentation.

Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin.

In this study, in order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in the high pullulan producer Aureobasidium melanogenum P16 isolated from the mangrove ecosystem. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain EI36, one of the transformants, produced 40.92 U/ml of inulinase activity while its wild-type strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27 %) of the inulinase produced by the recombinant strain EI36 was secreted into the culture. During the 10-l fermentation, 70.57 ± 1.3 g/l of pullulan in the fermented medium was attained from inulin (138.0 g/l) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase, and most of the reducing sugars were used by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.

The mechanism of improved pullulan production by nitrogen limitation in batch culture of Aureobasidium pullulans

Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation.

Understanding the effect of interaction among aeration, agitation and impeller positions on mass transfer during pullulan fermentation by Aureobasidium pullulans

Pullulan is a non-ionic, water-soluble homopolysaccharide producedviafermentation usingAureobasidium pullulans, a black yeast.

Simultaneously enhanced production and molecular weight of pullulan using a two‐stage agitation speed control strategy

AbstractBACKGROUNDPullulan with high molecular weight (Mw) is attractive for industrial application. Previous studies indicated that high yield and high Mw of pullulan cannot be simultaneously obtained under constant agitation speeds or constant dissolved oxygen levels during batch pullulan fermentation. In this study, a two‐stage agitation speed control strategy was developed to obtain pullulan with both high yield and high Mw.RESULTSBatch pullulan production at various constant agitation speeds ranging from 200 to 600 rpm was carried out in a 5 L stirred tank fermentor. Based on kinetic analysis of batch processes, activity assays of the enzymes associated with pullulan biosynthesis and biodegradation, and intracellular energy supply under different constant agitation speeds, a two‐stage control strategy that shifted the agitation speed from 500 to 400 rpm at 18 h proved to effectively achieve simultaneous high yield of high Mw pullulan.CONCLUSIONA maximum pullulan concentration of 27.4 g L−1 and final Mw of 3.09 × 106 Da were obtained using the two‐stage agitation speed control strategy, which were both greatly improved compared with those obtained at constant agitation speeds. © 2014 Society of Chemical Industry