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Enhanced production of Ca²⁺-polymalate (PMA) with high molecular mass by Aureobasidium pullulans var. pullulans MCW.

Wang YK, Chi Z, Zhou HX, Liu GL, Chi ZM - Microb. Cell Fact. (2015)

Bottom Line: The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca(2+)-PMA production.During 10-L batch fermentation, 152.52 g/L of Ca(2+)-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium.Mw (the apparent molecular weight) of the purified PMA was 2.054 × 10(5) (g/moL) and the purified PMA was estimated to be composed of 1784 L-malic acids.

View Article: PubMed Central - PubMed

Affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, 266003, China. w84234102@sina.com.

ABSTRACT

Background: Polymalic acid (PMA) has many applications in food and medical industries. However, so far it has not been commercially produced by fermentation. Therefore, it is very important how to develop an economical process for a large scale production of PMA by one step fermentation.

Results: After over 200 strains of Aureobasidium spp. isolated from the mangrove systems in the South of China were screened for their ability to produce Ca(2+)-polymalate (PMA), it was found that Aureobasidium pullulans var. pullulans MCW strain among them could produce high level of Ca(2+)-PMA. The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca(2+)-PMA production. Then, 121.3 g/L of Ca(2+)-PMA was produced by A. pullulans var. pullulans MCW strain within 120 h at flask level. During 10-L batch fermentation, 152.52 g/L of Ca(2+)-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium. After purification of the Ca(2+)-PMA from the culture and acid hydrolysis of the purified Ca(2+)-PMA, HPLC analysis showed that A. pullulans var. pullulans MCW strain produced only one main component of Ca(2+)-PMA and the hydrolysate of the purified Ca(2+)-PMA was mainly composed of L-malic acid. Mw (the apparent molecular weight) of the purified PMA was 2.054 × 10(5) (g/moL) and the purified PMA was estimated to be composed of 1784 L-malic acids.

Conclusions: It was found that A. pullulans var. pullulans MCW strain obtained in this study could yield 152.52 g/L of Ca(2+)-PMA within the short time, the produced PMA had the highest molecular weight and the medium for production of Ca(2+)- PMA by this yeast was very simple.

No MeSH data available.


Related in: MedlinePlus

Effects of different concentrations of CaCO3 (a) and glucose (b) on Ca2+-PMA production (grey) and cell growth (black) by the yeast strain MCW. Data are given as mean ± SD, n = 3.
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Fig2: Effects of different concentrations of CaCO3 (a) and glucose (b) on Ca2+-PMA production (grey) and cell growth (black) by the yeast strain MCW. Data are given as mean ± SD, n = 3.

Mentions: It has been well documented that a high initial C/N ratio (nitrogen starvation) and the presence of CaCO3 in the medium are required to boost l-malic acid biosynthesis for PMA production in microbial cells [11, 14]. Therefore, it is very important to optimize the glucose and CaCO3 concentrations in the Ca2+-PMA production medium. So the effects of different concentrations of glucose and CaCO3 on Ca2+-PMA production and cell growth by the yeast strain MCW were examined as described in “Methods”. The results in Fig. 2b showed that when the Ca2+-PMA production medium contained 140.0 g/L glucose, the amount of the Ca2+-PMA in the culture reached the highest (121.11 g/L) while the results in Fig. 2a showed that when the Ca2+-PMA production medium contained 65.0 g/L of CaCO3, the amount of the Ca2+-PMA in the culture was the highest (121.13 g/L). The results in Fig. 2 also revealed that the Ca2+-PMA production by the yeast strain MCW was closely related to its cell growth. The data of the Ca2+-PMA titers and cell mass obtained above were subjected to One-way Analysis of Variance (ANOVA) [20]. P values were calculated by Student’s t test (n = 3). P values less than 0.05 were considered statistically significant. The statistical analysis was performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). The results demonstrated that there were big differences between the Ca2+-PMA titers and cell mass shown in Fig. 2. It has been reported that during the nitrogen starvation (a high initial C/N ratio in the production medium), expression of most of the glycolytic genes and all the genes related to the cytosolic l-malic acid production pathway in Aspergillus oryzae were highly upregulated [21]. It also has been evidenced that CaCO3 is necessary for l-malate production in the fermentation medium because the presence of CaCO3 in the medium can keep pH constant of around 6.5 and provide CO2 as a substrate for efficient production of l-malate, the precursor of PMA biosynthesis [14, 22]. The results in Fig. 2 were indeed consistent the finding. It was thought that in the nitrogen starvation, many zinc finger proteins such as Msn2/4, Gat1, Gln3 and AreA are involved in biosynthesis of organic acids, single cell oils, exopolysaccharides, antibiotics, toxins and others in fungal cells [23, 24, 28]. However, it is still completely unknown if such zinc finger proteins could be involved in PMA biosynthesis in A. pullulans var. pullulans MCW strain used in this study. Such investigation is being carried out in this laboratory.Fig. 2


Enhanced production of Ca²⁺-polymalate (PMA) with high molecular mass by Aureobasidium pullulans var. pullulans MCW.

Wang YK, Chi Z, Zhou HX, Liu GL, Chi ZM - Microb. Cell Fact. (2015)

Effects of different concentrations of CaCO3 (a) and glucose (b) on Ca2+-PMA production (grey) and cell growth (black) by the yeast strain MCW. Data are given as mean ± SD, n = 3.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528779&req=5

Fig2: Effects of different concentrations of CaCO3 (a) and glucose (b) on Ca2+-PMA production (grey) and cell growth (black) by the yeast strain MCW. Data are given as mean ± SD, n = 3.
Mentions: It has been well documented that a high initial C/N ratio (nitrogen starvation) and the presence of CaCO3 in the medium are required to boost l-malic acid biosynthesis for PMA production in microbial cells [11, 14]. Therefore, it is very important to optimize the glucose and CaCO3 concentrations in the Ca2+-PMA production medium. So the effects of different concentrations of glucose and CaCO3 on Ca2+-PMA production and cell growth by the yeast strain MCW were examined as described in “Methods”. The results in Fig. 2b showed that when the Ca2+-PMA production medium contained 140.0 g/L glucose, the amount of the Ca2+-PMA in the culture reached the highest (121.11 g/L) while the results in Fig. 2a showed that when the Ca2+-PMA production medium contained 65.0 g/L of CaCO3, the amount of the Ca2+-PMA in the culture was the highest (121.13 g/L). The results in Fig. 2 also revealed that the Ca2+-PMA production by the yeast strain MCW was closely related to its cell growth. The data of the Ca2+-PMA titers and cell mass obtained above were subjected to One-way Analysis of Variance (ANOVA) [20]. P values were calculated by Student’s t test (n = 3). P values less than 0.05 were considered statistically significant. The statistical analysis was performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). The results demonstrated that there were big differences between the Ca2+-PMA titers and cell mass shown in Fig. 2. It has been reported that during the nitrogen starvation (a high initial C/N ratio in the production medium), expression of most of the glycolytic genes and all the genes related to the cytosolic l-malic acid production pathway in Aspergillus oryzae were highly upregulated [21]. It also has been evidenced that CaCO3 is necessary for l-malate production in the fermentation medium because the presence of CaCO3 in the medium can keep pH constant of around 6.5 and provide CO2 as a substrate for efficient production of l-malate, the precursor of PMA biosynthesis [14, 22]. The results in Fig. 2 were indeed consistent the finding. It was thought that in the nitrogen starvation, many zinc finger proteins such as Msn2/4, Gat1, Gln3 and AreA are involved in biosynthesis of organic acids, single cell oils, exopolysaccharides, antibiotics, toxins and others in fungal cells [23, 24, 28]. However, it is still completely unknown if such zinc finger proteins could be involved in PMA biosynthesis in A. pullulans var. pullulans MCW strain used in this study. Such investigation is being carried out in this laboratory.Fig. 2

Bottom Line: The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca(2+)-PMA production.During 10-L batch fermentation, 152.52 g/L of Ca(2+)-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium.Mw (the apparent molecular weight) of the purified PMA was 2.054 × 10(5) (g/moL) and the purified PMA was estimated to be composed of 1784 L-malic acids.

View Article: PubMed Central - PubMed

Affiliation: College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, 266003, China. w84234102@sina.com.

ABSTRACT

Background: Polymalic acid (PMA) has many applications in food and medical industries. However, so far it has not been commercially produced by fermentation. Therefore, it is very important how to develop an economical process for a large scale production of PMA by one step fermentation.

Results: After over 200 strains of Aureobasidium spp. isolated from the mangrove systems in the South of China were screened for their ability to produce Ca(2+)-polymalate (PMA), it was found that Aureobasidium pullulans var. pullulans MCW strain among them could produce high level of Ca(2+)-PMA. The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca(2+)-PMA production. Then, 121.3 g/L of Ca(2+)-PMA was produced by A. pullulans var. pullulans MCW strain within 120 h at flask level. During 10-L batch fermentation, 152.52 g/L of Ca(2+)-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium. After purification of the Ca(2+)-PMA from the culture and acid hydrolysis of the purified Ca(2+)-PMA, HPLC analysis showed that A. pullulans var. pullulans MCW strain produced only one main component of Ca(2+)-PMA and the hydrolysate of the purified Ca(2+)-PMA was mainly composed of L-malic acid. Mw (the apparent molecular weight) of the purified PMA was 2.054 × 10(5) (g/moL) and the purified PMA was estimated to be composed of 1784 L-malic acids.

Conclusions: It was found that A. pullulans var. pullulans MCW strain obtained in this study could yield 152.52 g/L of Ca(2+)-PMA within the short time, the produced PMA had the highest molecular weight and the medium for production of Ca(2+)- PMA by this yeast was very simple.

No MeSH data available.


Related in: MedlinePlus