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.

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.

Introduction: Glutamate decarboxylase is a class Ⅱ amino acid decarboxylase dependent onpyridoxal-5′-phosphate (PLP), which catalyzes the decarboxylation of substrateL-glutamate (L-Glu) to synthesize γ-aminobutyric acid (GABA). The low activity ofglutamic acid decarboxylase (GAD) and its ability to catalyze only under acidicconditions limit its application in biosynthesis of GABA.Methods: Taking glutamic acid decarboxylase from Lactobacillus plantarum, which produces GABA, as the research object, the mutation site was determined by amino acid sequence analysis of GAD, the mutation was introduced by primers, and the mutant was constructed by whole plasmid PCR and expressed in Escherichia coli. Then, the enzymatic properties of the mutant were analyzed. Finally, the three-dimensional structure of the mutant was simulated to support the experimental results.Results and Discussion: In this case, mutants E313S and Q347H of glutamate decarboxylase from L. pltarum LC84 (LpGAD) were constructed by targeted mutagenesis. Compared with the wild-type, their enzyme activity increased by 62.4% and 12.0% at the optimum pH 4.8, respectively. In the range of pH 4.0–7.0, their enzyme activity was higher than that of the wild-type, and enzyme activity of mutant E313S was 5 times that of the wild-type at pH 6.2. Visualization software PyMOL analyzed the 3D structure of the mutant predicted by homologous modeling, and the results showed that mutant E313S may broadened the reaction pH of LpGAD through the influence of surface charge, while mutant Q347H may broadened the reaction pH of LpGAD through the stacking effect of aromatic rings. In a word, mutants E313S and Q347H were improved the enzyme activity and were broadened the reaction pH of the enzyme, which made it possible for it to be applied in food industry and laid the foundation for the industrial production of GABA.