Optimization of the process parameters for reduction of gossypol levels in cottonseed meal by functional recombinant NADPH-cytochrome P450 reductase and cytochrome P450 CYP9A12 of Helicoverpa armigera

Cheng Chen,Wen-Ju Zhang,Yan Zhang,Wenhui Pi,Wenting Yang,Cunxi Nie,Xi Ma,Jing Liang

doi:10.1186/s13568-019-0823-4

Abstract

Gossypol is a toxic polyphenolic product that is derived from cotton plants. The toxicity of gossypol has limited the utilization of cottonseed meal (CSM) in the feed industry. The gene, Helicoverpa armigera CYP9A12, is a gossypol-inducible cytochrome P450 gene. The objective of our study was to obtain the functional recombinant H. armigera CYP9A12 enzyme in Pichia pastoris and to verify whether this candidate enzyme could decrease gossypol in vitro. Free and total gossypol contents were detected in the enzyme solution and in CSM. The H. armigera CYP9A12 enzyme degraded free concentration of gossypol. After optimization of the single-test and response surface method, free gossypol content could be decreased to 40.91 mg/kg in CSM by the H. armigera CYP9A12 enzyme when the initial temperature was 35 °C, the enzymatic hydrolysis time lasted 2.5 h, the enzyme addition was 2.5 mL, and the substrate moisture was 39%.

Highlights

The presence of toxic free gossypol in cottonseed meal (CSM) greatly limited its efficient use in animal feed (Matlin and Zhou 1984; Matlin et al 1988; Yildirimaksoy et al 2004)
To validate the detoxification effect of H. armigera cytochrome P450-monooxygenase CYP9A12 (CYP9A12) in the enzyme reaction solution, the free and total gossypol contents were determined in the control group, endogenous group, and H. armigera CYP9A12 enzyme group, respectively
The ability of the H. armigera CYP9A12 enzyme to degrade gossypol was defined as the amount of one micromole of gossypol degraded per minute catalyzed by 1 mg of enzyme at Enzyme reaction solution

Summary

Introduction

The presence of toxic free gossypol in cottonseed meal (CSM) greatly limited its efficient use in animal feed (Matlin and Zhou 1984; Matlin et al 1988; Yildirimaksoy et al 2004). Our previous studies demonstrated that microbial detoxification of CSM can effectively eliminate its toxic effect (Zhang et al 2006a, The induction of the H. armigera P450 monooxygenase CYP6AE14 and CYP9A12 genes (Mao et al 2007; Celorio-Mancera et al 2011; Zhou et al 2010) were possibly involved in the resistance and metabolism of gossypol (Jia et al 2008; Kong et al 2010; Krempl et al 2016a, b). The NADPH-cytochrome P450 reductase (CPR) was essential to help cytochrome P450 monooxygenase detoxify the substrates and xenobiotics (Guengerich et al 2009) because the coexpression of house fly NADPH P450 reductase with H. armigera CYP6AE14 (Tao et al 2012) and CYP6AE14 microsomes (Krempl et al 2016a) resulted in epoxidation activity towards aldrin

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Conclusion