首页|Cloning and heterologous expression of subtilisin SAPN, a serine alkaline protease from Melghiribacillw thermohalophilus Nari2A~T in Escherichia coli and Pichia pastoris

Cloning and heterologous expression of subtilisin SAPN, a serine alkaline protease from Melghiribacillw thermohalophilus Nari2A~T in Escherichia coli and Pichia pastoris

扫码查看
The sapN gene, encoding the extracellular subtilisin SAPN, a serine alkaline protease from Melghiribacillus thermohalophilus Nari2AT, was isolated, sequenced, and heterologously expressed in Escherichia coli BL21(DE3) pLysS using pUT57 and pTrc99A vectors and in E. coli BL21-AITM using the GatewayTM pDESTTM 17 vector. Conversely, three cassettes encoding pre-pro-subtilisin (rSAPN/SP-Pro-M), pro-subtilisin (rSAPN/Pro-M), and the mature-subtilisin (rSAPN/M) were hyperexpressed in Pichia pastoris SMD1168 and X33 using the pPICZ?C vector. rSAPNs were purified, characterized, and compared to wild-type SAPN. The deduced amino acid sequences exhibited high similarity with subtilisins from Bacillus strains. The highest sequence identity (96 %) was observed with the Bacillus licheniformis MP1 protease, with a 10-residue difference. Compared to SAPN and untagged rSAPNs, (His)6-tagged enzymes showed the highest activity and stability at alkaline pH and high temperature, the highest hydrolysis degree on crab and shrimp by-products, and the best catalytic efficiency. It was found that His6-rSAPN/SP-Pro-Ms expressed in P. pastoris strains was more active than those produced in E. coli. To initiate structure-function relationships, a 3D-model of the Pro-SAPN was built based on the available structures of common subtilisins. These data constitute a pivotal first step toward the creation of new efficient rSAPNs with enhanced catalytic properties and high potential for biotechnological and industrial uses.

Melghiribacillus thermohalophilusSubtilisinExpressionPpastorisComparative modeling

Mechri, Sondes、Jaouadi, Nadia Zarai、Bouacem, Khelifa、Allala, Fawzi、Bouraoui, Aicha、Ferard, Celine、Rekik, Hatem、Noiriel, Alexandre、Abousalham, Abdelkarim、Bouanane-Darenfed, Amel、Hacene, Hocine、Lederer, Florence、Baciou, Laura、Jaouadi, Bassem

展开 >

Univ Sfax, Ctr Biotechnol Sfax CBS, Lab Biotechnol Microbienne Enzymat & Biomol LBMEB, Route Sidi Mansour Km 6,BP 1177, Sfax 3018, Tunisia

Univ Sci & Technol Houari Boumediene USTHB, Fac Sci Biol FSB, Lab Biol Cellulaire & Mol LBCM, Equipe Microbiol, BP 32, Bab Ezzouar 16111, Alger, Algeria

Univ Paris Saclay, Inst Chim Phys ICP, UMR 8000, CNRS, Bat 350,15,Ave Jean Perrin, F-91405 Orsay, France

Univ Lyon, Inst Chim & Biochim Mol & Supramol ICBMS, Univ Lyon 1, UMR 5246,CNRS,Genie Enzymat Membranes Biomimet &, Bat Raulin,43 Bd 11 Novembre 1918, F-69622 Villeurbanne, France

展开 >

2021

Process biochemistry

Process biochemistry

EISCI
ISSN:1359-5113
年,卷(期):2021.105(Jun.)
  • 6
  • 42