3.1 Whole-cell catalysis process of primary diols oxidized by G. oxydans
The dual functional modules of hydroxyl acids give them the application prospects in many high-end fields. However, the traditional technology for hydroxyl acid preparation not only releases a large amount of waste, but also produces serious by-product. Hence, employing G. oxydansas the core catalyst to explore green and environmentally friendly preparation process of hydroxyl acids is an effective approach to solve the current industry bottlenecks. In order to explore the reaction mechanism of G. oxydans whole-cell catalyzing linear primary diols, five diols were selected as substrates for kinetic study with OD600=2.
As shown in Figure 1, the bioprocesses for catalysis of EG, 1,3-PG and 1,4-BG (100 mmol/L) byG. oxydans were described respectively. As shown in the figure, the final products of EG, 1,3-PG and 1,4-BG could only be catalyzed to glycolic acid (GA), 3-HPA and 4-hydroxybutyric acid (4-HBA), respectively, but could not be further catalyzed to diacids. Moreover, the average consumption rates of EG, 1,3-PG and 1,4-BG were 10.89, 20.4, 24.8 mmol/L/h, respectively. Compared with chemical method, the catalytic rate and purity of the products are satisfactory. Therefore, the results confirm that G. oxydans has a promising future in the production of hydroxyl acids (C2-C4) from primary diols. In addition, Figure 1 also shows the bioprocess for the catalysis of 1,5-PG and 1,6-HG (100 mmol/L) by G. oxydans respectively. Surprisingly, when the methylene number was ≥ 5, the primary diol reaction processes showed qualitative differences. As shown in the figure, 1,5-PG and 1,6-HG catalysis followed a step-by-step process, with further catalysis to glutaric acid (GTA) and adipic acid (AA) when primary diols were oxidized to hydroxyl acids. Moreover, in terms of catalytic rate, the catalytic processes of 1,5-PG and 1,6-HG were very similar. The substrate consumption time for 100 mmol/L were less than 3 h and 4 h, while the average substrate consumption rates reached 31.22 and 45.50 mmol/L/h, respectively. Apparently, although the reaction efficiency of C5/C6 was excellent, the product quality was not satisfactory due to the byproducts. However, at present, hydroxyl acid is receiving much research attention in materials science, due to its excellent properties of two functional modules. Hence, we need to further explore the regulation of cheap substrates primary diols oxidized by G. oxydans to lay a theoretical foundation for finding the technology of selective regulation to prepare hydroxyl acids form primary diols.
3.2 Selective regulation for hydroxyl acid production from C5 diols by pH control
Previous studies show that the carbon chain length of the substrate directly determines the product type of whole-cell catalysis. When C≥5, the primary diols are oxidized to form intermediate hydroxyl acids, which are further converted to diacids. Nowadays, in the current organic acid industry, the preparation of diacids has been approaching maturity and there are cheaper and more convenient production methods. Nevertheless, there are still many bottlenecks in the industrial preparation of hydroxyl acids, such as low yield, impure products, toxic raw materials, high cost and other fatal defects. In 2019, Keiichi et al employ over supported platinum catalysts only obtain 62% yield of target products including 5-HVA, δ-valerolactone, and methyl 5-hydroxyvalerate(Asano et al., 2019). The result not only has a low yield, but also contains abundant derivatives, which seriously affects the purity of the products. Moreover, in 2021, Hee et al fermentative production of 5‑HVA by metabolically engineered Corynebacterium glutamicum . Finally, about 55 g/L 5-HVA and 10 g/L GTA were produced during 28 h fermentation [18]. Therefore, targeted regulate of the process for selective catalysis of high-grade primary diols (C≥5) is a promising way to develop efficient and green hydroxyl acid industrial preparation.
As shown in Figure 2, we conducted the whole-cell catalysis of 1,5-PG under different pH gradients, including pH=2.5, 3.5, 4.5, 5.5, 6,5. Surprisingly, the results showed that when pH≥5.5, GAT would not be produced even if the substrate 1,5-PG completely bio-oxidized to 5-HVA. However, when pH is less than 5.5, the whole-cell catalysis would show two-stage reactions, that is, 1,5-PG would generate 5-HVA in the first step, and then 5-HVA would be catalyzed to GTA. It is noteworthy that the result under pH=2.5 was contrary to the law we obtained, because it is difficult for cells to maintain normal physiological activity under extremely acidic conditions, and G. oxydans has lose catalytic ability after 2 h.
In order to verify the accuracy of pH regulation, we further used the entire cells as the crude enzyme, and measure the enzyme activity of 1,5-PG and 5-HVA as substrates by employing the microplate reader. The enzyme activity was calculated by Formula 1, and the results are shown in Table 1. From the results, we can clearly see that when 1,5-PG was employed as the substrate, the catalytic ability was stronger with the decrease of pH, which was similar with the previous whole-cell catalysis. As for 5-HVA as the substrate, when pH≥5.5, G. oxydans has no catalytic activity at all, which means that G. oxydans has no ability to transform 5-HVA to GTA under this condition. In summary, the results of 1,5-PG as the substrate indicated that the lower pH was, the higher catalytic efficiency would be. At the same time, when 5-HVA was used as substrate, the results indicated that the pH must exceed 5.5 to accurately control the product to 5-HVA. Therefore, the proposed scheme of pH-regulated whole-cell catalysis provided a green and high-quality process for the industrial production of 5-HVA. Finally, in order to directional obtain ultra-high titer of 5-HVA, we chose pH=5.5 to conduct bioreactor experiment.