Cloning and expression of ferulic acid esterase gene and its effect on wort filterability
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To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization.
Recombinant FaeA was purified from the fermentation broth, with the maximum specific activity of 48.4 ± 0.1 U mg−1. Adding it during mashing process for beer brewing raised the filtration rate by 14.5% while the turbidity and viscosity declined by 22 and 6.9%, respectively. Addition of FaeA increased the concentrations of free ferulic acid (FA) and arabinoxylan (AX) in the wort, while the polymeric arabinoxylans content declined significantly.
Recombinant FaeA was capable to prevent the oxidative gelation of PAX formation by breaking the cross-linking of FA among AX chains and improve the filtration performance of wort.
KeywordsArabinoxylan Ferulic acid esterase Oxidative gelation Pichia pastoris Wort filterability
This work was supported by the National High Technology Research and Development Program of China (2013AA102109), the National Natural Science Foundation of China (31701588), the Natural Science Foundation of Jiangsu Province, China (BK20170178), the Project Funded by the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.
Supplementary Table 1—Primers used in this study.
Supplementary Table 2—Optimal codons in Pichia pastoris.
Supplementary Table 3—Comparison of the expression and activity levels of FaeA in different strains.
Supplementary Figure 1—Alignment of nucleotide of the optimized and original Anfae A genes. Optimized sequences were designed using the GeMS software package and then synthesized by Sangon. Characters with shadow were the same nucleotides, and others were different.
Supplementary Figure 2—Screening of positive transformants with high expression of recombinant FaeA. The colonies were firstly cultured in buffered glycerol complex medium (1% yeast extract, 2% tryptone, 1.34% YNB, 1% glycerol, 4×10−5% biotin, and 100 mmol l-1 potassium phosphate, pH 6.0) with shaking at 30°C 220 rpm to reach OD600 2-6. The cells were then harvested via centrifugation and suspended in buffered methanol-complex medium (1% yeast extract, 2% tryptone, 1.34% YNB, 4×10−5% biotin, 1% methanol, and 100 mmol l-1 potassium phosphate, pH 6.0). After further culture for 96 h, 1% methanol was added every 24 h to induce the expression of recombinant FaeA. The supernatant of fermentation was added into the ethyl ferulate plates and positive transformants with highest activity of FaeA were selected according to the transparent circle diameter. The larger diameter of the transparent circle indicated higher FaeA activity.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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