High molecular weight β-poly(l-malic acid) produced by A. pullulans with Ca2+ added repeated batch culture
Introduction
β-Poly(malic acid) (PMLA) is a biodegradable, biocompatible and water soluble natural biopolyester with versatile pendant carboxy group, which allows the conjunction of biologically active molecules and/or a targeting moiety via appropriate chemical modifications [1], [2]. PMLA has attracted increasing attentions because of its potential application in medicine and other industries [3], [4]. It could be biosynthesized by the Physarum polycephalum and Aureobasidium pullulans. For P. polycephalum, the low concentration of PMLA (2.7 g/L) in broth [5] has restricted its further application for large-scale production of PMLA. While most strains of A. pullulans in genetically diverse phylogenetic clades could produce a high concentration of PMLA [6], [7], from 9.8 g/L [8] to 87.6 g/L [9] in free-cell fermentation with a stirred bioreactor. Therefore, A. pullulans was a promising microorganism for the industrial production of PMLA in the near future.
Besides the final concentration, PMLA molecular weight (Mw) is also an important factor affecting the PMLA applications. For example, PMLA with Mw of 5KDa could be used as a protease inhibitor [10], while PMLA with Mw of 50 KDa was used for synthesizing a new prototype of polymer-derived drug delivery system [11]. A minimal chain length of approximately 10 malate residues was required to inhibit homologous DNA polymerase α [12], and an intravenous PMLA nanobioconjugate (at least contain 8 malate residues) was used for inhibition of brain tumor growth [13]. Therefore, it is necessary to regulate PLMA Mw during PMLA biosynthesis. In this regard, different molecular weights of final PLMA products from 4.6 to 11 kDa by A. pullulans and Aureobasidium sp. strains [6], [8], [14], [15] were obtained under different fermentation conditions, implying that PLMA Mw can be controlled by optimizing the process parameters. However, there has no report regarding the variations of PMLA Mw during PMLA biosynthesis.
Although the available method for increase of PMLA Mw cannot be found in the present literature yet, Nagata et al. [14] reported that the addition of CaCO3 to culture media could increase the PMLA concentration, and the Mw of obtained PLMA was relatively high among the known results. Inspired by this, we hypothesize that CaCO3 addition during the fermentation can also increase the PLMA Mw. However, the addition of CaCO3 would produce various effects on the PLMA fermentation. For instance, when the experiment was carried out in flasks, the addition of CaCO3 would change both ionic strength and pH during the fermentation. When producing PMLA in bioreactor, the addition of CaCO3 as neutralizer could result in a highly viscous suspension because the cells interacted with the precipitates, and this would have a detrimental effect on the rate of oxygen transfer. Therefore, it is difficult to clarify the mechanisms how CaCO3 addition did affect the PMLA production and to find suitable fermentation conditions to regulate the Mw of PMLA products. To the best of our knowledge, the variation of PMLA Mw during the batch culture and the strategies to enhance PMLA Mw were not investigated yet.
In this work, different neutralizers were added during PMLA fermentations in order to seek the real reason influencing PMLA production (including PMLA concentration and Mw) with the addition of CaCO3, and then the relationship among PMLA production, PMLA molecular weight and cell growth was illustrated. The objective of this study is not only to clarify the effect of CaCO3 addition on PMLA production, but also to establish an approach to produce high Mw of PMLA by repeated batch culture.
Section snippets
Microorganism
A. pullulans ipe-1 (CGMCC No. 3337) used in this study was stored in China General Microbiological Culture Collection Center, Beijing, China. The strain was maintained on potato-glucose agar slant at 4 °C.
Culture medium
The compositions (w/v) of the seed culture medium were as follows: 8% glucose, 0.2% NaNO3, 0.01% KH2PO4, 0.02% MgSO4·7H2O, 0.05% KCl and 0.1% tryptone (LP0042, Oxoid LTD., Basingstoke Hampshire, England) in deionized water. The compositions of the fermentation medium in flasks were the same as
Unravelling the effect of CaCO3 addition on PMLA production
The effect of CaCO3 addition on cell growth and PMLA production is shown in Fig. 1. Without CaCO3 addition, the pH of the broth dropped to 3.99 because of the PMLA production. When CaCO3 was added in the broth, culture pH of the cultivations kept between 6.0 and 6.5. The highest PMLA production was obtained at 1.5% CaCO3 addition, while at above this CaCO3 concentration, PMLA production decreased. Moreover, it was found that with addition of CaCO3, the biomass slightly increased and then
Conclusion
In β-poly(l-malic acid) production by A. pullulans ipe-1, the Ca2+ addition contributed to the improvement of PMLA production at constant pH conditions, and the pH and Ca2+ were the two factors for promoting the PMLA concentration by CaCO3 addition. Further, adding exogenous Ca2+ in the broth of batch culture could enhance Mw of PMLA, and the molecular weight of PMLA increased from 12.522 to 18.693 kDa and then decreased in the late exponential growth phase. The Mw varied with Yp/x (g PMLA/g
Acknowledgements
The authors thank the National Science Foundation of China (No. 21406240), the Chinese Academy of Sciences’ Key Deployment Project (No. KSZD-EW-2-017-3) and the National High Technology Research and Development Program of China (No. 2014AA021005 and No. 2015AA021002) for the financial supports.
References (32)
- et al.
Nanoconjugate based on polymalic acid for tumor targeting
Chem. Biol. Interact.
(2008) - et al.
The optimization of polymalic acid peptide copolymers for endosomolytic drug delivery
Biomaterials
(2011) - et al.
Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly (β-l-malic acid)
J. Controlled Release
(2007) - et al.
Poly-(l)-malic acid: a new protease inhibitor from Penicillium cyclopium
Biochem. Biophys. Res. Commun.
(1969) - et al.
Nanoconjugate based on polymalic acid for tumor targeting
Chem-Biol. Interact.
(2008) Melanin production and differentiation in batch cultures of the polymorphic fungus Aureobasidium Pullulans
FEMS Microbiol. Lett.
(1980)- et al.
Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate
Bioresour. Technol.
(2008) - et al.
Effect of exogenous calcium on morphological development and biopolymer synthesis in the fungus Aureobasidium pullulans
Enzyme Microb. Technol.
(1997) - et al.
Poly (β-malic acid) production by the non-growing cells of Aureobasidium sp. strain A-91
J. Ferment. Bioeng.
(1996) - et al.
Biological and biosynthetic properties of poly-l-malate
FEMS Microbial. Lett.
(1992)
Production and degradation of β-Poly-l-malate in cultures of Physarum polycephalum
Cell Biol. Int. Reports
Bioresorbability and biocompatibility of aliphatic polyesters
J. Mater. Sci.-Mater. Med
Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum
Appl. Microbiol. Biotechnol.
Poly (β-l-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans
J. Ind. Microbiol. Biotechnol.
Overproduction of poly (β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system
Appl. Microbiol. Biotechnol.
Investigation of poly (β-l-malic acid) production by strains of Aureobasidium pullulans
Appl. Microbiol. Biotechnol.
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