Comparative Proteomic Analysis of Aureobasidium pullulans in the

Oct 7, 2014 - Department of Life Science, Hefei Normal University, Hefei 230061, ... and matrix-assisted laser desorption/ionization time-of-flight/ti...
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Comparative Proteomic Analysis of Aureobasidium pullulans in the Presence of High and Low Levels of Nitrogen Source Long Sheng,† Guilan Zhu,‡ and Qunyi Tong*,† †

The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China ‡ Department of Life Science, Hefei Normal University, Hefei 230061, China ABSTRACT: Pullulan, produced by Aureobasidium pullulans strain, has been broadly used in the food and medical industries. However, relatively little is known concerning the molecular basis of pullulan biosynthesis of this strain. In this paper, the effect of different concentrations of (NH4)2SO4 on pullulan fermentation was studied. Proteomics containing two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) were used to analyze the protein with different expressions of A. pullulans cells between the nitrogen limitation and nitrogen repletion. Maximum pullulan production reached 37.72 g/L when 0.6 g/L of initial (NH4)2SO4 was added. Excess nitrogen source would impel carbon flux flow toward biomass production, but decreased the pullulan production. Nitrogen limitation in A. pullulans seemed to influence the flux change of carbon flux flow toward exopolysaccharide accumulation. The findings indicated that 12 identified protein spots were involved in energy-generating enzymes, antioxidant-related enzymes, amino acid biosynthesis, glycogen biosynthesis, glycolysis, protein transport, and transcriptional regulation. These results presented more evidence of pullulan biosynthesis under nitrogen-limited environment, which would provide a molecular understanding of the physiological response of A. pullulans for optimizing the performance of industrial pullulan fermentation. KEYWORDS: Aureobasidium pullulans, pullulan, nitrogen limitation, proteomics

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INTRODUCTION Aureobasidium pullulans is generally known as the strain producing pullulan, an exocellular homopolysaccharide. Pullulan is broadly applied in the food and medical industries because of its prominent chemical and physical properties, such as low viscosity, nontoxicity, slow digestibility, high plasticity, and excellent film-forming.1 Pullulan is a linear homopolysaccharide composed of maltotriose reduplicative units connected by α-1,4-linkages. This particular linkage pattern confers pullulan with excellent solubility in water compared to other polysaccharides.2 Pullulan biosynthesis is a complex metabolic process under the control of environmental conditions. Factors known to affect pullulan production in A. pullulans involve the fungal strain used, carbon source, nitrogen source, incubation pH, culture temperature, dissolved oxygen level, and fermenter configuration.3 Bulmer et al. reported that the ammonium ion (NH4+) played an important part in pullulan formation.4 The exhaustion of nitrogen source is supposed to be a symbol for pullulan accumulation by A. pullulans,5 just like many other biopolymer-producing microorganisms, such as Azospirillum brasilense for the production of polyhydroxyalkanoates (PHA) and poly-3-hydroxybutyrate (PHB) or Agrobacterium sp. for curdlan production.6,7 Jiang et al. found that the optimum fermentation time for pullulan production and UDPGpyrophosphorylase activity was influenced by the nitrogen source in the culture.8 Complex protein mixture separation and protein identification can be solved by proteomics techniques, including 2-DE, in-gel digestion, and mass spectrometry. A large number of proteins, involved in morphological and biochemical process, © 2014 American Chemical Society

are qualitatively and quantitatively measured by proteomics, so as to provide exact analysis of alternative cell proteins in the course of growth, differentiation, and response to environmental factors.9−12 Although pullulan has been used in different fields in industry, the molecular basis of pullulan biosynthesis of A. pullulans is still poorly understood. To control the fermentation process, it is important to realize the occurrence and development in the cell at a molecular level. The objective of the present paper is to describe the comparative global protein profiles in A. pullulans cells in the presence of high and low levels of nitrogen source, thus extending our understanding of nitrogen metabolic regulation performance in relation to cell biomass growth and pullulan production.

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MATERIALS AND METHODS

Microorganism. A. pullulans CGMCC1234 was conserved on potato dextrose agar (PDA) at 4 °C and cultured every 2 weeks again. Seed Culture. The seed broth included 50.0 g of sucrose, 4.0 g of K2HPO4, 2.0 g of NaCl, 1.5 g of yeast extract, 0.8 g of (NH4)2SO4, and 0.2 g of MgSO4 in 1 L of distilled water. The medium pH was 6.5, and the medium was sterilized at 121 °C for 15 min. Culture Conditions. Cells were cultured on PDA at 28 °C for 96 h, then inoculated to a 250 mL flask containing 50 mL of the seed culture medium, and shaken at 28 °C and 200 rpm for 48 h. The pullulan fermentation was performed in a 5 L stirred tank fermentor (KF-5l, KoBioTech Bioengineering Equipment Co., Ltd.) with a Received: Revised: Accepted: Published: 10529

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working volume of 3 L of the production liquid medium including (g/ L) 80.0 g of sucrose, 6.0 g of K2HPO4, 0.9 g of yeast extract, 0.5 g of MgSO4, 4.0 g of NaCl, and different concentrations of (NH4)2SO4. The fermentor loaded with 3 L of the production culture medium was autoclaved at 121 °C for 15 min. A total of 150 mL of the seed culture medium was transferred to the production culture medium. The pullulan fermentation was performed at 28 °C and 300 rpm with an aeration rate of 3 L/min. The pH was maintained at 6.5 by the addition of either 2 M NaOH or 2 M HCl. Determination of Pullulan Production and Biomass. Properly distilled water was added to the fermentation liquor to dilute. The broth was heated at 80 °C in a water bath for 30 min and then centrifuged at 10000g for 20 min to separate the biomass and insoluble debris. Two volumes of precooling 95% (v/v) ethanol was added to the supernatant and stirred and placed at 4 °C for 24 h to deposit the exopolysaccharide thoroughly. The sediment was isolated by centrifugation at 15000g for 20 min, washed with ethanol again, and dried at 80 °C until the weight was constant. The biomass sediment from the initial centrifugation was washed three times with distilled water by centrifugation at 10000g for 5 min and dried at 80 °C until the dry cell weight (DCW) was constant. Pullulan production and DCW were expressed as grams per liter. (NH4)2SO4 Concentration. For residual (NH4)2SO4 concentration determination, a revised indophenol method was used.13 The fermentation liquor (2 mL) was centrifuged at 4000g to separate microorganisms and other precipitates. Hundred-fold distilled water was added to the supernatant to dilute and then adjusted to pH 7.0. Next, 25 mL of sample was mixed with 10 mL of phenol nitroprusside buffer. To this mixture was added 15 mL of hypochlorite reagent, and the mixture was held at room temperature for 45 min for color development. The absorbance of the solution was measured at 290 nm. Protein Preparation for 2-DE. Cells were harvested by centrifugation at 15000g for 20 min at 4 °C and washed three times with ultrapure water by centrifugation at 10000g for 5 min. Cells were ground in liquid nitrogen after freeze-drying. The fine powder was suspended in acetone including 0.07% (w/v) dithiothreitol (DTT) and 10% (w/v) trichloroacetic acid (TCA) and incubated at −20 °C overnight. The suspension was centrifuged at 15000g for 15 min at 4 °C, and the sediment was washed three times with precooling acetone including 0.07% (w/v) DTT. The sediment was dried and redissolved in rehydration solution [7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1.8 mM DTT, 0.5% (w/v) Pharmalyte 3−10] and then centrifuged at 15000g for 20 min at 4 °C. The limpid supernatant including the soluble proteins was stored in aliquots at −80 °C. The method of Bradford was used to determine protein concentration with bull serum albumin as standard.14 2-DE. The protein extracts (250 μg/gel) were loaded for 2-DE as follows. The 24 cm strips (Immobiline Drystrip) covering a nonlinear pH 3−11 (GE Healthcare) were rehydrated in 450 μL of rehydration solution for 16 h at room temperature. Isoelectric focusing was operated by the Ettan IPGphor (GE Healthcare) with a ceramic tray manifold at 20 °C. Each strip was limited to a maximum current of 50 μA. The isoelectric focusing electrophoresis program followed the protocol rehydration at 50 V for 12 h (step), 500 V for 1 h (step), 1000 V for 1 h (step), 10000 V for 1 h (gradient), and 11000 V for 10 h (step). The strip is taken from the strip holder. The redundant oil and protein solutions were absorbed by filter paper. The IPG gel strips were respectively incubated in an equilibration solution [50 mM TrisHCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.002% bromophenol blue] with 1% DTT and 2.5% iodoacetamide for 15 min. Then the equilibrated gel strips were put on a 1.5 mm 12.5% polyacrylamide gel and sealed with agarose sealing solution [25 mM Tris-base, 192 mM glycine, 0.1% (w/v) SDS, 0.5% agarose, and 0.002% (w/v) bromophenol blue]. The second-dimensional separation was performed by the ETTAN DALTsix (GE Healthcare) in a cold chamber at 15 °C. SDS-PAGE was operated at a constant voltage of 100 V for 45 min and at 200 V for 6−8 h until the bromophenol

blue arrived at the bottom of the gel. After SDS-PAGE, the separated proteins were visualized by silver staining as previously described.15 Image Analysis. Silver-stained gels were scanned using an Image Scanner (Amersham Biosciences). Date analysis was carried out utilizing Image Master 2D Platinum 5.0 software (Amersham Biosciences). For each treatment, three gels from three independent treatments were studied. Gel spots were automatically tested and manually edited and deleted when necessary. Evidently spots were marked by calibrating and matching the gel. Next, the three replicate gels were compared to each other and merged into a master gel, containing the spots distributed on the three gels for every independent experiment. To quantify and contradistinguish the protein spots, standardization of the volume ratio was performed. The significantly changed spots were chosen with at lowest a 0.2-fold decrease and a 5.0-fold increase in the normalized volume ratio. In-Gel Digestion. The differentially expressed protein spots were excised for digestion. The gel spot was washed and the destained solution removed. Fifty percent acetonitrile (ACN) and 100% ACN were successively added for 5 min. The samples were rehydrated in 2− 4 μL of mass spectroscopy grade gold trypsin (Promega, Madison, WI, USA) solution (20 μg/mL in 25 mM NH4HCO3) for 30 min. Then, 20 μL of cover solution (25 mM NH4HCO3, 10% ACN) was added for digestion for 16 h at 37 °C. The supernatant was shifted to another tube, and the gel was extracted with 50 μL of extraction buffer [67% ACN and 5% trifluoroacetic acid (TFA)]. The supernatant of the gel spot and the peptide extracts were combined and freeze-dried for mass spectrometry. Mass Spectrometry Analysis. The freeze-dried power was suspended with 5 μL of 0.1% TFA and then mixed with a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50% ACN and 0.1% TFA, at the ratio of 1:1. One microliter of the mixture was spotted on a stainless steel sample target board. Peptide MS and MS/ MS were performed on an ABI 5800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster City, CA, USA). Data were obtained in a positive MS reflector using a CalMix5 standard to adjust the instrument (ABI5800 Calibration Mixture). The integrated MS and MS/MS data were disposed by the GPS Explorer V3.6 software (Applied Biosystems, USA) with default parameters. On the basis of the combination of MS and MS/MS spectra, proteins were successfully identified in the standard of 95% or higher confidence interval of their scores in the Mascot V2.3 search engine (Matrix Science Ltd., London, UK), using the search parameters as follows: NCBInr-Funji database; trypsin as the digestion enzyme; one missed cleavage site; fixed modifications, carbamidomethyl (C); partial modifications, acetyl (protein N-term), deamidated (NQ), dioxidation (W), oxidation (M); 100 ppm for precursor ion tolerance and 0.5 Da for fragment ion tolerance.

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RESULTS AND DISCUSSION Effect of Different (NH4)2SO4 Concentrations on Pullulan Fermentation. The effect of various concentrations of (NH4)2SO4 on cell growth and pullulan production by A. pullulans CGMCC1234 is shown in Figure 1. The results acquired at different (NH4)2SO4 concentrations proved that the (NH4)2SO4 played a vital role on pullulan formation. The carbon flux from merging into fungal cell material to pullulan synthesis was dependent on (NH4)2SO4 concentration. When 0.6 g/L of (NH4)2SO4 was added into the medium, pullulan concentration achieved the maximum and the production was 37.72 g/L. A higher concentration of (NH4)2SO4 brought about the higher level of biomass but reduced the pullulan production. Just 9.45 g/L of pullulan was obtained when 1.5 g/ L (NH4)2SO4 was added; meanwhile, 12.31g/L DCW was obtained. The possible reason was that excess (NH4)2SO4 could not be entirely expended until the end of the fermentation stage, which led to the growth of cell biomass and reduced pullulan production. However, insufficient nitro10530

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Figure 1. Effect of (NH4)2SO4 concentration on DCW and pullulan production of A. pullulans CGMCC1234 during culture development in a 5 L stirred tank fermentor. Values are given as means ± standard deviation (n = 3).

gen source sparked off the reduction of both biomass and pullulan. When 0.3 g/L (NH4)2SO4 was added to the culture medium, just 20.03 g/L pullulan was obtained. Comparison of the fermentation parameters of A. pullulans CGMCC1234 with 0.6 and 1.2 g/L (NH4)2SO4 is shown in Figure 2. With 0.6 g/L (NH4)2SO4 addition, the biomass growth was apparently inhibited after 36 h. In the meantime, (NH4)2SO4 was consumed almost at 36 h. Nevertheless, 1.2 g/ L (NH4)2SO4 was exhausted until 84 h and the biomass constantly increased through the end of the fermentation. Interestingly, compared to the 1.2 g/L (NH4)2SO4 group, the pullulan formation of the 0.6 g/L (NH4)2SO4 group was more quickly increased during the 4 days of cultivation. The production of pullulan under nitrogen limit was 3.3-fold as compared to the nitrogen-replete group. Providing excess nitrogen could induce the promotion of cell biomass, but not increase pullulan production.16 Nitrogen metabolism was a strictly conducted process in fungi; therefore, fungi had the capacity to exploit finite nitrogen source for cell growth. Effective supervision of the nitrogen metabolism process possibly endowed fungi with the ability to utilize varieties of ecological niches.17 For A. pullulans CGMCC1234 in the nitrogen-limited condition, the medium with 0.6 g/L (NH4)2SO4 group displayed sensitivity to pullulan formation. Excess nitrogen source would impel carbon flux flow toward biomass production. The depletion of nitrogen induced exopolysaccharide accumulation of A. pullulans fermentation. Protein Identification. In previous studies, we found that exopolysaccharide accumulation was not tightly related to cell growth. When A. pullulans cells reached a stationary phase and the cell growth was limited, pullulan could be synthesized more as a secondary metabolite.18 To search for differences in physiological metabolism and its administration between the cell biomass growth and secondary metabolite pullulan biosynthesis in A. pullulans CGMCC1234 cells in the presence of high and low levels of nitrogen source, proteins extracted from the cells of culture medium with 0.6 and 1.2 g/L (NH4)2SO4 group at 60 h were subjected to proteome analysis. The representational gel maps of the proteins separated from the A. pullulans cells cultivated in the nitrogen-limited medium and the nitrogen-replete medium are shown in Figure 3. As

Figure 2. Effect of nitrogen-limited cultivation with 0.6 g/L (NH 4 ) 2 SO 4 and nitrogen-replete cultivation with 1.2 g/L (NH4)2SO4 on the DCW (a), pullulan production (b), and residual (NH4)2SO4 concentration (c) of A. pullulans CGMCC1234 during culture development in a 5 L stirred tank fermentor. Values are given as means ± standard deviation (n = 3).

analyzed through Image Master, approximately 1100 visualized spots were found on the gels. After comparison and analysis the gel images, altogether 19 protein spots from the whole cell protein gels were excised and subjected to MALDI-TOF/TOF. The successfully identified proteins were marked on the 2-DE gel maps (Figure 3), and more detailed information about identified proteins is shown in Table 1. Among the 19 protein spots cut for MALDI-TOF/ TOF analysis, 12 protein spots were successfully identified, achieving a 63% identification rate. Meanwhile, the identified proteins were matched to known proteins from the fungus species and other microorganisms. Of the 12 protein spots identified, 7 were up-regulated under the nitrogen repletion and 5 were up-regulated under the nitrogen limitation (Table 1). The up-regulated proteins in the nitrogen-replete sample contained nascent polypeptide-associated complex subunit β, peroxiredoxin-6, α-glucan branching enzyme, fructose-bisphos10531

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Figure 3. Comparative 2-DE gel analyses of cellular proteins of A. pullulans CGMCC1234 cultivated in the nitrogen-limited medium (a) and the nitrogen-replete medium (b). Protein spots marked on the maps with arrows and numbers represent the 12 identified proteins analyzed with MALDI-TOF/TOF.

Table 1. Identification of Differentially Expressed Protein Spots from A. pullulans CGMCC1234 Cells Produced with the Addition of 0.6 and 1.2 g/L (NH4)2SO4 by Proteomic Analysis with 2D-GE and MALDI-TOF/TOF MSa spot no.

accession no.

1 2 3 4 5 6

gi|485919582 gi|334683219 gi|336260705 gi|145249406 gi|225681491 gi|302420083

7

gi|189202520

putative cyanate hydratase protein mitochondrial peroxiredoxin-1 hypothetical protein SMAC_07435 ATP synthase subunit β Hsp70-like protein nascent polypeptide-associated complex subunit β peroxiredoxin-6

8 9 10 11 12

gi|320586098 gi|398408315 gi|225681690 gi|154323902 gi|302501588

α-glucan branching enzyme fructose-bisphosphate aldolase arginase enolase hypothetical protein ARB_01037

mascot score

coverage (%)

theor Mw

theor pI

Neof usicoccum parvum UCRNP2 Diplodia seriata Sordaria macrospora k-hell Aspergillus niger CBS 513.88 Paracoccidioides brasiliensis Pb03 Verticillium alfalfae VaMs.102

115 247 56 56 163 94

8 21 13 9 4 7

17443 25596 25156 19969 73805 17253

5.96 8.59 5.97 4.75 5.91 4.96

Pyrenophora tritici-repentis Pt-1CBFP Grosmannia clavigera kw1407 Zymoseptoria tritici IPO323 Paracoccidioides brasiliensis Pb03 Botryotinia f uckeliana B05.10 Arthroderma benhamiae CBS 112371

157

9

25367

5.88

278 80 118 97 103

5 4 6 3 4

133585 39652 31748 47196 45451

5.65 5.63 5.46 5.26 5.18

protein name

organism

a The 19 chosen protein spots showed significant difference in expression level between the nitrogen-limited and the nitrogen-replete conditions of culture as analyzed by 2D eletrophoresis. Of the 19 protein spots excised for MALDI-TOF/TOF analysis, 12 proteins were identified.

transcriptional regulation of glycolysis flux. For instance, enolase participated in adjusting and controlling glycolysis flux through induction of the expression of Rag1 glucose permease in Kluyveromyces lactis.21 It was obvious that more (NH4)2SO4 up-regulated fructose-bisphosphate aldolase and enolase protein, leading to the carbon flux toward glycolic pathway for the growth of biomass. α-Glucan branching enzyme (spot 8 in Figure 3 and Table 1) was an essential enzyme that catalyzed the development of the α-1,6-glucosidic linkages in glucan chains by scission of an α1,4-linked oligosaccharide from growing α-1,4-glucan chains and the subsequent attachment of the oligosaccharide to the α1,6 position. It was involved in the biosynthesis of both glycogen and capsular α-D-glucan.22 Excess nitrogen source might accelerate the biosynthesis of intracellular glycogen but reduce the pullulan production. Simon et al. found that pullulan synthesis was increasing while glycogen production was decreasing during growth, so it should be possible that the kinetics of production of these two kinds of polysaccharides were inversely correlated.23 Arginase (spot 10 in Figure 3 and Table 1) was a manganesecontaining enzyme and ubiquitous to all domains of life. Arginase catalyzed the hydrolysis of L-arginine into L-ornithine and urea. This reaction was the initial step in catabolism of

phate aldolase, arginase, enolase, and hypothetical protein ARB_01037. In comparison, five identified proteins, including putative cyanate hydratase protein, mitochondrial peroxiredoxin-1, hypothetical protein SMAC_07435, ATP synthase subunit β, and Hsp70-like protein, revealed up-regulation in the nitrogen-limited sample. Proteome Changes. Fructose-bisphosphate aldolase (spot 9 in Figure 3 and Table 1) and enolase (spot 11 in Figure 3 and Table 1) were two proteins involved in the glycolytic pathway. Fructose-bisphosphate aldolase, generally referred to as aldolase, was an enzyme responsible for the catalysis of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). Aldolase could also catalyze sedoheptulose 1,7-bisphosphate and fructose 1phosphate to generate DHAP.19 Glycolysis, a catabolic pathway, used the forward reaction. Enolase, also called phosphopyruvate hydratase, was a metalloenzyme catalyzing a reversible reaction of the conversion of phosphoenolpyruvate (PEP) to 2-phosphoglycerate (2-PG), the penultimate step of glycolytic pathway. Aldolase and enolase both belonged to the class lyase. These two enzymes could catalyze the reverse reaction, according to the environmental concentrations of substrates.20 Increasing evidence has revealed that enolase had mutiple capabilities and played an important part in the 10532

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arginine and the terminal step in anabolism of urea.24 Excess nitrogen source stimulated amino acid biosynthesis and metabolism in A. pullulans CGMCC1234 cells. Nascent polypeptide-associated complex subunit β (spot 6 in Figure 3 and Table 1) was part of the nascent polypeptideassociated complex (NAC). A component of the NAC was a dynamic ribosomal transport corridor. It protected the newly formed polypeptides from interaction with other cytoplasmic proteins to guarantee suitable nascent protein targeting. Furthermore, the NAC could effectively enhance the interaction of ribosome and the membrane of mitochondria and also prevent improper interaction of nonsecretory nascent polypeptides translated from ribosome and translocation sites in the endoplasmic reticulum membrane. Hence, the NAC could efficiently improve the import of mitochondrial protein.25−27 Numerous proteins were synthesized when A. pullulan cells rapidly multiplied in sufficient nitrogen source. The up-regulation of nascent polypeptide-associated complex was conducive to protein transport and transcription regulation. The 70 kDa heat shock proteins (Hsp70) (spot 5 in Figure 3 and Table 1) belonged to a family of widespread heat shock proteins. Proteins with similar structures were found in almost all organisms. Hsp70 played a crucial role in protein folding in the cells and meanwhile helped to protect the cells from environmental pressure. Hsp70 seemed to be involved in treating defective and damaged proteins. Hsp70 could be able to protect protein from aggregating through close connection to fractional synthesized peptide sequences.28 As the nitrogen source was consumed, the environment outside the cells became rigorous; the expression of more Hsp70 could protect A. pullulans cells from oxidative stress, which generally hurts proteins, leading to aggregation and partial unfolding. ATP synthases (spot 4 in Figure 3 and Table 1) were membrane-bound enzymes existing in mitochondrial inner membrane and vacuoles. The basic function of ATP synthases was to synthesize ATP using an electrochemical ion gradient. Some ATP synthases worked in reverse, using the energy from the hydrolysis of ATP to create a proton gradient. Studies suggested that pullulan synthesis involved UDP-glucose, required ATP, and proceeded through lipid intermediates.29 It was obvious that nitrogen-limited cells generated more ATP necessary for metabolism including polymerization of precursor to form pullulan and excretion of pullulan to the culture broth. In this work, the fermentation results obtained showed that proper concentration (0.6 g/L) of (NH4)2SO4 was important for pullulan biosynthesis. Excess nitrogen source would enhance the accumulation of biomass, but decreased the pullulan production. Nitrogen limitation in A. pullulans CGMCC1234 seemed to influence the flux change of carbon flux flow toward exopolysaccharide accumulation. The proteomic results revealed that the expression of antioxidantrelated enzymes and energy-generating enzymes and the depression of the enzymes concerning amino acid biosynthesis, glycogen biosynthesis, glycolysis, protein transport, and transcriptional regulation under nitrogen limitation, caused a conversion of metabolic flux from the glycolysis pathway to the pullulan biosynthesis pathway. This research supplies a new perspective into the understanding of the mechanism of pullulan biosynthesis and will apply practical significance for the improvement of pullulan production from A. pullulans.

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AUTHOR INFORMATION

Corresponding Author

*(Q.T.) Phone/fax: +86 510 85919170. E-mail: qytong@263. net. Funding

This work was supported by the National Natural Science Foundation of China (31401657), the Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes

The authors declare no competing financial interest.

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REFERENCES

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf503390f | J. Agric. Food Chem. 2014, 62, 10529−10534