Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from <i>Exophiala aquamarina</i>

doi:10.13523/j.cb.2105018

China Biotechnology

2021, Vol. 41

Issue (10): 19-27 DOI: 10.13523/j.cb.2105018

Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from Exophiala aquamarina

LIANG Ai-ling^1,²,LIU Wen-ting^1,²,WU Pan^1,²,LI Qian²,GAO Jian²,ZHANG Jie²,LIU Wei-dong²,JIA Shi-ru^1,^**(

),ZHENG Ying-ying^2,^**(

)

1 Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
2 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

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Abstract

Objective: In order to promote the enzymatic complete detoxification of zearalenone and the application in feed industry, we proposed to develop a new hydrolase exhibiting higher activity to α-ZOL. Methods: A novel zearalenone hydrolase EaZHD sequence from Exophiala aquamarina CBS 119918 with a total length of 263 amino acids was obtained. The recombinant plasmid pET46-Eazhd was constructed and expressed in E. coli. Then EaZHD was purified with Ni-NTA affinity chromatography and DEAE ion exchange column. The enzymatic properties and activity analysis were evaluated with HPLC. Results: The results showed that the activity of EaZHD was 0.764 U/mg against ZEN and 1.529 U/mg against α-ZOL, which is 2-fold higher. The optimal pH of EaZHD was 8.6 and the optimal temperature was 40 °C. It had a better stability at alkaline pH. The thermal stability is better than ZHD101. Three amino acid residues around active site were identified to play a key role in the catalysis activity and the other two residues were found to affect the substrate specificity. Conclusion: The study provides the basis for the enzymatic detoxification of zearalenone in the feed and stock raising industries.

Key words： Novel zearalenone hydrolase α-Zearalenol Enzymatic properties Enzymatic detoxification

Received: 10 May 2021 Published: 08 November 2021

ZTFLH:

Q819

Corresponding Authors: Shi-ru JIA,Ying-ying ZHENG E-mail: jiashiru@tust.edu.cn;zheng_yy@tib.cas.cn

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	Ai-ling LIANG
	Wen-ting LIU
	Pan WU
	Qian LI
	Jian GAO
	Jie ZHANG
	Wei-dong LIU
	Shi-ru JIA
	Ying-ying ZHENG

Cite this article:

LIANG Ai-ling,LIU Wen-ting,WU Pan,LI Qian,GAO Jian,ZHANG Jie,LIU Wei-dong,JIA Shi-ru,ZHENG Ying-ying. Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from Exophiala aquamarina. China Biotechnology, 2021, 41(10): 19-27.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2105018 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I10/19

Fig.1 Sequence alignment of EaZHD and other homologous proteins

Table 1 Primer list

Fig.2 The PCR result of Eazhd

Fig.3 The SDS-PAGE analysis of EaZHD Lane 1: The broken supernatant of BL21(DE3)(pET46) strain; Lane 2-5: BL21(DE3)(pET46-Eazhd) before induction; induced with IPTG; purification with the His Trap TM HP column; purification with the DEAE ion exchange column

Fig.4 The activities of EaZHD and other homologous proteins

Fig.5 Effect of pH on EaZHD activity (a) and stability (b)

Fig.6 Effect of temperature on EaZHD activity (a) and stability (b)

Fig.7 The superimposition of modeled structure of EaZHD with ZHD101 (a)The color scheme is green for EaZHD and purple for ZHD101 in panel (b)The color scheme for the ligands and amino acid residues are the same as those described in panel. The catalytic triad is also shown, colored blue. Dashed lines indicate hydrogen bonding

Fig.8 Chemical structures of ZEN and α-ZOL (a) Describes the hydrolytic process of ZEN, showing the structures of the substrate (left), intermediate (middle), and product (right) (b)Structures of α-ZOL are also shown

Fig.9 The activity of EaZHD mutants

Fig.10 The structural comparsion of EaZHD wild type and mutants (a)-(f) The structural superposition between wild-type and mutants D31A, D31H, H125A, V127H, N153H and V154H, respectively


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