High molecular weight β-poly(l-malic acid) produced by A. pullulans with Ca²⁺ added repeated batch culture

Abstract

β-Poly(malic acid) (PMLA) has attracted increasing attentions because of its potential application in medicine and other industries. In this study, the variation of PMLA molecular weight (M_w) in the batch culture and the strategies to enhance PMLA M_w were studied. Adding exogenous Ca²⁺ (0.1 g/L CaCl₂) to the medium caused a significant increase in both PMLA concentration and M_w (11.38% and 26.3%, respectively) when Na₂CO₃ was used as the neutralizer. The M_w of PMLA during the process of batch culture, which associated with the specific PMLA production per unit cell mass (Y_p/x) before glucose was depleted, increased from 12.522KDa to its maximum 18.693KDa and then kept decreasing until the end of the culture. Compared with the results in batch culture, M_w increased by 84.4% (up to 19.51 kDa) with a productivity of 1.1 g h⁻¹ L⁻¹ when the cells were maintained in exponential growth phase during Ca²⁺ added repeated batch culture. The present work provides an efficient approach to obtain superior quality PMLA product with high M_w.

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 (M_w) is also an important factor affecting the PMLA applications. For example, PMLA with M_w of 5KDa could be used as a protease inhibitor [10], while PMLA with M_w 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 M_w 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 M_w can be controlled by optimizing the process parameters. However, there has no report regarding the variations of PMLA M_w during PMLA biosynthesis.

Although the available method for increase of PMLA M_w 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 M_w 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 M_w. 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 M_w of PMLA products. To the best of our knowledge, the variation of PMLA M_w during the batch culture and the strategies to enhance PMLA M_w 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 M_w) 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 M_w of PMLA by repeated batch culture.