Abstract
Hydrolytic activity for chlorogenic acid (CGA) has been recognized as an important side activity of some types of ferulic acid esterases. The purpose of this work was to enhance the efficient expression of ferulic acid esterase (FAE) and to explore its application in the processing of sunflower seed. Two novel FAEs from Aspergillus aculeatus (AaSD14) were expressed in genetically engineered E. coli BL21 (DE3), and their properties, including temperature, pH, metal ions and substrate specificity, were characterized after purification. Competitive CGA hydrolysis activity was observed in these recombined ferulic acid esterases (reFAEs) with reFAE1 of 246.37 U/g and reFAE2 of 340.95 U/g, which were 56.6 and 78.4 times higher than that of the wild strain (4.35 U/g), respectively. Meanwhile, the fermentation cycle was greatly shortened to 2.0 d. These reFAEs were recognized as type C FAE through substrate specificity assays. Treatment of sunflower seed protein (SSP) using reFAE2 resulted in a remarkable color change, from green to milk-white, confirming the activity of CGA biodegradation. Therefore, it shows certain potential in the processing of sunflower seed and other related foodstuffs.
Highlights
Sunflower seeds are an important source of oil, and a high-quality source of protein, which have the advantages of few anti-nutritional factors, low allergenicity, reasonable and balanced amino acid (AA) composition, etc
The sequence analysis revealed that there were two open reading frames (ORFs), which could both encode the ferulic acid esterase (FAE). These two ORFs consisted of 1500 bp and 1569 bp, encoding 500 and 523 amino acids (AAs), respectively
The deduced amino acid sequence of recombined ferulic acid esterases (reFAEs) was used to perform a BLAST search in the National Center for Biotechnology Information (NCBI) and SwissProt databases. It revealed a relative similarity between reFAEs and other FAEs, including alignment of the amino acid sequences among reFAE1, reFAE2, and other FAEs: AofaeB from Aspergillus oryzae (Genbank: PDB: 3WMT_B) [20] (28% and 32% identity, respectively), AnFaeB from Aspergillus niger (Genbank: UniProtKB/SwissProt: Q8WZI8.1) [21] (29% and 30% identity, respectively), AN1772.2 from Aspergillus nidulans FGSC A4 (Genbank: UniProtKB/Swiss-Prot: Q5B2G3.1) [22] (28% and 32% identity, respectively), and FofaeC from Fusarium oxysporum (GenBank: SCN69328.1) [23] (29% and 31% identity, respectively)
Summary
Sunflower seeds are an important source of oil, and a high-quality source of protein, which have the advantages of few anti-nutritional factors, low allergenicity, reasonable and balanced amino acid (AA) composition, etc. Under high pH conditions, the oxidatively polymerized CGA dimers tend to covalently bind to the polar groups of the protein, thereby generating green quinones [3] and affecting the sensory quality, functional properties and nutritional value of SSP [4,5,6]. This is the reason why SSP has been regarded as waste or low valuable animal feed, leading to financial loss and low utilization. The CGA content in sunflower seeds can reach 71.4% of the total phenolic content in sunflower seeds and play a critically negative role in protein quality
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