Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum.
- Author:
Ruihua LÜ
1
;
Linna SHI
1
;
Xirong ZHANG
1
;
Zhao FENG
1
Author Information
- Publication Type:Journal Article
- Keywords: Sclerotinia sclerotiorum; cutinase; nitrophenyl butyrate; prokaryotic expression; protein activity
- MeSH: Ascomycota/genetics*; Carboxylic Ester Hydrolases; Cloning, Molecular; Phylogeny
- From: Chinese Journal of Biotechnology 2022;38(1):386-395
- CountryChina
- Language:Chinese
- Abstract: Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.
