Cloning and expression of ferulic acid esterase gene and its effect on wort filterability

  • Dianhui Wu
  • Guolin Cai
  • Xiaomin Li
  • Bing Li
  • Jian Lu
Original Research Paper
  • 30 Downloads

Abstract

Objectives

To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization.

Results

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.

Conclusions

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.

Keywords

Arabinoxylan Ferulic acid esterase Oxidative gelation Pichia pastoris Wort filterability 

Notes

Acknowledgements

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.

Supporting information

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.

Supplementary material

10529_2018_2511_MOESM1_ESM.tif (13.7 mb)
Supplementary material 1 (TIFF 14051 kb). Supplementary Fig. 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
10529_2018_2511_MOESM2_ESM.tif (15.4 mb)
Supplementary material 2 (TIFF 15745 kb). Supplementary Fig. 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 a 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 the highest activity of FaeA were selected according to the transparent circle diameter. The larger diameter of the transparent circle indicated higher FaeA activity
10529_2018_2511_MOESM3_ESM.doc (46 kb)
Supplementary material 3 (DOC 46 kb)
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Supplementary material 4 (DOC 54 kb)
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Supplementary material 5 (DOC 29 kb)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education; National Engineering Laboratory for Cereal Fermentation Technology, School of BiotechnologyJiangnan UniversityWuxiChina

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