Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties

被引：7

作者：

Shettar, Shreya S. ^{[1
]}

Bagewadi, Zabin K. ^{[1
]}

Yaraguppi, Deepak A. ^{[1
]}

Das, Simita ^{[2
]}

Mahanta, Nilkamal ^{[2
]}

Singh, Surya P. ^{[3
]}

Katti, Aditi ^{[1
]}

Saikia, Dimple ^{[3
]}

机构：

[1] KLE Technol Univ, Dept Biotechnol, Hubballi 580031, Karnataka, India

[2] Indian Inst Technol, Dept Chem, Dharwad 580011, Karnataka, India

[3] Indian Inst Technol Dharwad, Dept Biosci & Bioengn, Dharwad 580011, Karnataka, India

来源：

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES | 2023年 / 249卷

关键词：

Subtilisin; Cloning; Expression; Purification; Biological properties; Analytical characterization; SERINE ALKALINE PROTEASE; BIOCHEMICAL-CHARACTERIZATION; PURIFICATION; CLONING; PROTEINS; DESIGN;

D O I：

10.1016/j.ijbiomac.2023.125960

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5 alpha cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified similar to 40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 mu g/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC50 of 5 mu M and 12 mu M respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries.

引用

页数：26

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