Functional cell surface display of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis using N-acetylmuraminidase as the anchoring motif.
- Author:
Xiaohua LI
1
;
Xinfeng HUANG
;
Xiaohu SHAO
;
Lin LI
Author Information
1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- Publication Type:Journal Article
- MeSH:
Electroporation;
Endo-1,3(4)-beta-Glucanase;
genetics;
metabolism;
Escherichia coli;
genetics;
metabolism;
Glycoside Hydrolases;
genetics;
metabolism;
Green Fluorescent Proteins;
genetics;
metabolism;
Lactococcus lactis;
enzymology;
genetics;
Recombinant Fusion Proteins;
genetics;
metabolism;
Recombination, Genetic
- From:
Chinese Journal of Biotechnology
2009;25(1):89-94
- CountryChina
- Language:Chinese
-
Abstract:
In this report, we utilized N-Acetylmuraminidase (AcmA) to develop a whole-cell catalyst of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis. The PCR-amplified full-length acmA gene from L. lactis MB191 was fused with the green fluorescent gene (gfp), followed by ligating the chimeric acmA-gfp into the Escherichia coli-L. lactis shuttle expression vector pMG36k, yielding the recombinant plasmid pMB137. SDS-PAGE analysis showed that the constitutive expression of AcmA-GFP fusion protein in the L. lactis AS1.2829 construct harboring pMB137 (named MB137), with the predicted Mr of 74 kD. Western blotting, GFP specific fluorescence intensity assays and flow cytometry analysis confirmed that AcmA-GFP was immobilized on the outer membrane, which constituted approx. 35% of the total intracellular fusion protein. Furthermore, acmA was fused with a PCR-amplified encoding fragment of the endo-beta-1, 3-1, 4-glucanase gene (gls) from Bacillus sublitis BF7658, resulting in the recombinant plasmid pMB138. By transferring pMB138 into L. lactis AS1.2829, the derived L. lactis MB138 expressing the AcmA-GLS fusion enzyme exhibited a distinct whole-cell glucanase activity (by 12 U/mL) compared to the control strain, indicating AcmA had served as a functional anchoring motif to immobilize the heterologous enzyme on the cell surface of L. lactis.
