Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000

Xylan polymer backbone can be metabolized by xylanase enzyme. Xylanase has been widely studied for its biological function in food industry and paper pulp bleaching. Metabolising xylan using xylanase is the preferred approach as it is the easiest and cheapest method available. Xylanase has bee...

Full description

Saved in:

Bibliographic Details
Main Author:	Roslan, Abdullah Munir
Format:	Thesis
Language:	English
Published:	2017
Subjects:	Xylanases Lactococcus lactis Gene expression
Online Access:	http://psasir.upm.edu.my/id/eprint/90373/1/FBSB%202019%2026%20ir.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-upm-ir.90373
record_format	uketd_dc
spelling	my-upm-ir.903732021-12-01T06:21:57Z Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000 2017-04 Roslan, Abdullah Munir Xylan polymer backbone can be metabolized by xylanase enzyme. Xylanase has been widely studied for its biological function in food industry and paper pulp bleaching. Metabolising xylan using xylanase is the preferred approach as it is the easiest and cheapest method available. Xylanase has been cloned and overproduced in both Gram negative Escherichia coli and Gram positive Bacillus subtilis. Although B. subtilis and E. coli are known cell factories for expression of heterologous proteins, the production of xylanase in these hosts eventually reduces the quality of the products with the presence of impurities such as proteases and endotoxins. Therefore, the production of xylanase using Lactococcus lactis is a safer strategy to produce xylanase due to its food-grade status. This study was aimed to develop a genetically modified Lactococcus lactis NZ9000 strain harbouring a plasmid that secretes Bacillus coagulans ST-6 endoxylanase into the exterior environment. Recombinant clones developed in this study was found to successfully secrete the xylanase using its native signal peptide, L. lactis NZ9000 secreted endogenous protein, USP45 signal peptide and Pediococcus pentosaceus K1 signal peptide; SPK1. Secreted xylanase from transformant colonies was detected by the clear zone formations on remazol-brilliant-blue-xylan (RBB-xylan)-containing agar plate assay after 24 h incubation. The expression of this enzyme in the transformants was further confirmed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western immunoblotting analysis. A band size of ~20 kDa corresponding to B. coagulans ST-6 xylanase was identified in the samples of His�Tagged purified protein derived from affinity chromatography of the culture media of induced recombinant clones. The use of different signal peptides showed differences in the secretion of xylanase. Signal peptide SPK1 used for L. lactis NZ9000 has higher secretion efficiency of 25.3% compared to the other two signal peptides; USP45 (19.9%) and NSP (18.2%). However, the recombinant strain using signal peptide USP45 produced and secreted the highest amount of xylanase (16.9 µg/mL) among all three strains Xylanases Lactococcus lactis Gene expression 2017-04 Thesis http://psasir.upm.edu.my/id/eprint/90373/ http://psasir.upm.edu.my/id/eprint/90373/1/FBSB%202019%2026%20ir.pdf text en public masters Universiti Putra Malaysia Xylanases Lactococcus lactis Gene expression
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Xylanases Lactococcus lactis Gene expression
spellingShingle	Xylanases Lactococcus lactis Gene expression Roslan, Abdullah Munir Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
description	Xylan polymer backbone can be metabolized by xylanase enzyme. Xylanase has been widely studied for its biological function in food industry and paper pulp bleaching. Metabolising xylan using xylanase is the preferred approach as it is the easiest and cheapest method available. Xylanase has been cloned and overproduced in both Gram negative Escherichia coli and Gram positive Bacillus subtilis. Although B. subtilis and E. coli are known cell factories for expression of heterologous proteins, the production of xylanase in these hosts eventually reduces the quality of the products with the presence of impurities such as proteases and endotoxins. Therefore, the production of xylanase using Lactococcus lactis is a safer strategy to produce xylanase due to its food-grade status. This study was aimed to develop a genetically modified Lactococcus lactis NZ9000 strain harbouring a plasmid that secretes Bacillus coagulans ST-6 endoxylanase into the exterior environment. Recombinant clones developed in this study was found to successfully secrete the xylanase using its native signal peptide, L. lactis NZ9000 secreted endogenous protein, USP45 signal peptide and Pediococcus pentosaceus K1 signal peptide; SPK1. Secreted xylanase from transformant colonies was detected by the clear zone formations on remazol-brilliant-blue-xylan (RBB-xylan)-containing agar plate assay after 24 h incubation. The expression of this enzyme in the transformants was further confirmed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western immunoblotting analysis. A band size of ~20 kDa corresponding to B. coagulans ST-6 xylanase was identified in the samples of His�Tagged purified protein derived from affinity chromatography of the culture media of induced recombinant clones. The use of different signal peptides showed differences in the secretion of xylanase. Signal peptide SPK1 used for L. lactis NZ9000 has higher secretion efficiency of 25.3% compared to the other two signal peptides; USP45 (19.9%) and NSP (18.2%). However, the recombinant strain using signal peptide USP45 produced and secreted the highest amount of xylanase (16.9 µg/mL) among all three strains
format	Thesis
qualification_level	Master's degree
author	Roslan, Abdullah Munir
author_facet	Roslan, Abdullah Munir
author_sort	Roslan, Abdullah Munir
title	Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
title_short	Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
title_full	Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
title_fullStr	Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
title_full_unstemmed	Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000
title_sort	heterologous expression and secretion of bacillus coagulans st-6 xylanase in lactococcus lactis nz9000
granting_institution	Universiti Putra Malaysia
publishDate	2017
url	http://psasir.upm.edu.my/id/eprint/90373/1/FBSB%202019%2026%20ir.pdf
_version_	1747813617591386112

Heterologous expression and secretion of Bacillus coagulans ST-6 xylanase in Lactococcus lactis NZ9000

Similar Items