Efficient Secretory Expression and Purification on Three Insoluble Amidohydrolases for Ochratoxin A Hydrolysis by Pichia pastoris

• Published in: Journal of agricultural and food chemistry 2024-07, Vol.72 (29), p.16403-16411
• Main Authors: Zhang, Xuanjun, Ma, Xue, Dai, Guangqing, Fu, Xiaojie, Zhou, Yu
• Format: Article
• Language: English
• Subjects: Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - metabolism; Biotechnology and Biological Transformations; Escherichia coli - genetics; Escherichia coli - metabolism; Fermentation; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression; Hydrolysis; Kinetics; Ochratoxins - chemistry; Ochratoxins - metabolism; Pichia - genetics; Pichia - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomycetales - enzymology; Saccharomycetales - genetics; Saccharomycetales - metabolism
• ISSN: 0021-8561; 1520-5118; 1520-5118
• DOI: 10.1021/acs.jafc.4c03804

As a highly toxic mycotoxin, ochratoxin A (OTA) is widely contaminating agricultural products and has various toxicological effects. Bioenzymes for OTA degradation have shown promising potential for detoxification. Other than the efficient amidohydrolase ADH3 previously, two novel amidohydrolases ADH1 and AMD3 were obtained in this study. During Escherichia coli expression, the expressed protein solubility was very low and will limit future industrial application. Here, high copy number integrations were screened, and the amidohydrolases were efficiently secretory expressed by Pichia pastoris GS115. The protein yields from 1.0 L of fermentation supernatant were 53.5 mg for ADH1, 89.15 mg for ADH3, and 79.5 mg for AMD3. The catalytic efficiency (K cat/K m) of secretory proteins was 124.95 s–1 mM–1 for ADH3, 123.21 s–1 mM–1 for ADH1, and 371.99 s–1 mM–1 for AMD3. In comparison to E. coli expression, the active protein yields substantially increased 15.78–51.53 times. Meanwhile, two novel amidohydrolases (ADH1 and AMD3) showed much higher activity than ADH3 that produced by secretory expression.