Expression and application of aflatoxin degrading enzyme gene in Pichia pastoris

Abstract

In this study, three aflatoxin degrading enzyme genes, tv‐adtz, arm‐adtz and cu‐adtz, were heterologously expressed in Pichia pastoris. The protein expression of the enzyme solution was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the results showed that specific protein bands were detected and the target genes were successfully integrated into Pichia pastoris. The enzyme activities and detoxification efficiency of TV‐ADTZ, Arm‐ADTZ and Cu‐ADTZ crude enzyme solutions were detected, and the highest enzyme activities were up to 3.57 U/mL, 4.30 U/mL and 2.41 U/mL, and the highest degradation rates were up to 45.58%, 60.0% and 34.21%, respectively. Arm‐ADTZ with the best degradation effect was selected and designed for detoxification application experiments to test its detoxification efficiency of AFB1 in aqueous phase and in the process of moldy ground corn and preparation of DDGS, respectively, and the degradation rates reached 78.94%, 56.48% and 24.31% after 24 h of reaction, respectively. Thus, it can be seen that the aflatoxin‐degrading enzyme gene was successfully integrated into Pichia pastoris and secreted for expression, and the expressed product could effectively degrade AFB1.This article is protected by copyright. All rights reserved

Recombinant Oxidase from Armillaria tabescens as a Potential Tool for Aflatoxin B1 Degradation in Contaminated Cereal Grain

“…To date, a wide range of recombinant enzymes able to attack mycotoxins have been produced using prokaryotic or eukaryotic expression systems [ 20 , 45 , 47 , 49 , 50 , 51 , 52 ]; for some of them, their decontaminating activity has been experimentally confirmed on grain and other food/feed products [ 45 , 49 , 51 ]. For example, a high decontamination potential was recently demonstrated for one recombinant AFO (Arm-ADTZ) in experiments on the enzymatic treatment of mold on corn (56.48% degradation within 24 h) and grain by-products obtained in the course of the distillation process in ethanol production [ 45 ].…”

“…Its crystal structure and the nucleotide sequence of a gene encoding a full-length protein have been determined [ 53 ], the safety of the resulting AFB1 degradation products has been confirmed [ 27 ], an additional dipeptidyl peptidase activity has been revealed [ 53 ], and at least three functional enzymes heterologously expressed in P. pastoris G115 have been reported [ 47 , 48 ]. Some kinetic characteristics, such as the K m value, the specific activity, and the half-life period at 30–50 °C were described for one of these recombinant AFOs [ 45 , 47 ], as well as for an AFO isolated from A. tabescens [ 34 , 41 ]. In our study, the use of a new signaling peptide promoting effective AFO secretion allowed us to prevent a partial protein degradation observed in the previous experiments with the AFO-containing cultural liquid of transformed yeast cells [ 48 ] and to provide a sufficient production of the extracellular enzyme possessing the target activity.…”

“…However, their development requires an available technology of AFO production and secretion that can be achieved via the use of a system for a heterologous expression of a recombinant enzyme. A few earlier reports [45][46][47] as well as our preliminary study [48] on recombinant AFO production indicated the possibility of using transformed Pichia pastoris strains as the appropriate heterologous producers of this enzyme. The authors of these in vitro studies demonstrated the ability of the obtained recombinant AFOs to degrade AFB1 and determined some kinetic characteristics and the target activity level of the enzymes at different pH levels and temperatures [45][46][47][48].…”

High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

“…[ 30 ] At the same time, P. pastoris distinguishes itself as an advantageous host for recombinant hCPB1 expression due to precise regulation with methanol promoters, [ 31 ] efficient extracellular secretion of proteins, well‐established molecular tools, high genetic stability, and capabilities for high‐density fermentation. [ 32–34 ]…”

“…[30] At the same time, P. pastoris distinguishes itself as an advantageous host for recombinant hCPB1 expression due to precise regulation with methanol promoters, [31] efficient extracellular secretion of proteins, well-established molecular tools, high genetic stability, and capabilities for high-density fermentation. [32][33][34] In this study, to construct P. pastoris microbial cell factory for recombinant hCPB1 production, we investigated the factors impeding the secretion of hCPB1 and implemented CRISPR-mediated gene deletion to engineer the conventional protein secretion pathway, thereby reducing the mis-sorting of hCPB1 to the vacuole. To enhance the specific activity of hCPB1, we employed evolutionary and coevolution analyses to pinpoint hotspots that potentially regulate its catalytic activities.…”