Xian Zhang

Feruloyl esterase (EC3.1.1.73; FAE) can degrade biomass to release ferulic acid (FA), which has a high application in bioprocessing, food, pharmaceutical, paper, feed, and other industrial fields. A strain of Klebsiella oxytoca Z28 with ferulic esterase activity was screened from Daqu. In addition, the FAE gene was expressed in Escherichia coli BL21 (DE3). The enzyme consists of 340 amino acids with a molecular mass of 37.7 kDa. The FAE enzyme activity was 463 U/L when the substrate was ethyl 4-hydroxy-3-methoxycinnamate and the optimum temperature and pH were 50 °C and 8.0, respectively. The enzyme had good stability at temperatures of 25–40 °C and a pH of 8.0. Ba²⁺, Cu²⁺, Mn²⁺, and Ca²⁺ had a strong inhibitory effect on the enzyme activity, and Na⁺ had a promotive effect on the enzyme activity. The de-starching wheat bran was degraded by KoFAE, and the FA release was up to 227.15 µg/g. This indicated that the heterologous expression of KoFAE from Klebsiella oxytoca Z28 in E. coli had a certain potential of biodegradation, which can be applied to the degradation of agricultural waste to obtain high value-added FA products.

Polyethylene (PE) is widely used, and it has caused serious environmental problems due to its difficult degradation. At present, the mechanism of PE degradation by microorganisms is not clear, and the related enzymes of PE degradation need to be further explored. In this study, Acinetobacter baumannii Rd-H2 was obtained from Rhizopertha dominica, which had certain degradation effect on PE plastic. The degradation performance of the strains was evaluated by weight loss rate, SEM, ATR/FTIR, WCA, and GPC. The multi-copper oxidase gene abMco, which may be one of the key genes for PE degradation, was analyzed and successfully expressed in E. coli. The laccase activity of the gene was determined, and the enzyme activity was up to 159.82 U/L. The optimum temperature and pH of the enzyme are 45 °C and 4.5 respectively. It shows good stability at 30–45 °C. Cu²⁺ can activate the enzyme. The abMCO was used to degrade polyethylene film, showing a good degradation effect, proving that the enzyme could be the key to degrading PE.

Feruloyl esterase (FAE)-producing micro-organisms to obtain ferulic acid (FA) from natural substrates have good industrial prospects, and the synergistic effect of multiple bacteria can better improve the yield of FA. In this study, on the premise of the synergistic effect of FAE, hemicellulose, and cellulase, the key strain Klebsiella oxytoca Z28 with FAE was combined with CMCase and Xylanase-producing strains to produce FA. The combination of strains with higher FA production are Klebsiella oxytoca Z28, Klebsiella pneumoniae JZE, Bacillus velezensis G1, and their FA production can reach 109.67 μg/g, which is 15% higher than that of single bacteria. To explore the effects of temperature, Ph, inoculum amount, distillers grains concentration and fermentation time on the FAE activity of the combination of strains in the fermentation process, and determined that temperature, Ph, and fermentation time were the main influencing factors and optimized through orthogonal design. The optimized fermentation conditions are 34 °C, Ph 8.0, and fermentation days for 6 days, the FAE activity can reach 270.78 U/L, and the FA yield of the combined strain is 324.50 μg/g, which is 200% higher than that of single-strain fermentation.