Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease s...

Full description

Saved in:
Bibliographic Details
Published in:Journal of microbiology and biotechnology 2020-11, Vol.30 (11), p.1720-1728
Main Authors: Yao, Zhuang, Meng, Yu, Le, Huong Giang, Lee, Se Jin, Jeon, Hye Sung, Yoo, Ji Yeon, Kim, Hyun-Jin, Kim, Jeong Hwan
Format: Article
Language:Korean
Subjects:
aprE
Bacillus subtilis
fibrinolytic activity
vpr
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40oC, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.
ISSN:1017-7825
1738-8872