Secretion of Bacillus Hydrolytic Enzymes using Food-grade Expression System Montarop Yamabhai1*, Peenida Namvijitra1, Suttipong Sak-Ubol1, Dietmar Haltrich2 1School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand 2Laboratory of Food Biotechnology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria Beta-mannanase, ManB Chitosanase, Csn Food-grade expression systems • Bacillus subtilis • E. coli K12 • Lactic acid bacteria (LAB) – NICE system : Lactococcus lactis – pSIP-based system : Lactobacillus spp. • pSIP409, pSIP609 • Lactobacillus plantarum Lactobacillus plantarum Expression System • Food-grade bacteria • Generally recognized as safe status (GRAS) • Probiotics • No endotoxins • No inclusion bodies • Simple cultivation, non-aerated fermentation • Complex nutritional requirements • Acidifying culture, required pHcontrol pSIP-based expression system Diep, D.B., Mathiesen, G., Eijsink, V.G., and Nes, I.F. (2009). Use of lactobacilli and their pheromonebased regulatory mechanism in gene expression and drug delivery. Curr Pharm Biotechnol 10, 62-73. • quorum-sensing mechanisms to induce gene expression, using bacteriocins (Sakacin A and Sakacin P). • Suitable for Lactobacillus spp. • Promoter PsapA in the pSIP300 series and PsppA or PsppQ (ProfX) in the pSIP400 series • All components are located in one plasmid with modular design Using Alanine Racemase (alr) for Food-grade expression alr D-alanine is a component of the peptidoglycan structure of bacterial cell wall Chitosanse (Csn) Chitooligosaccharides (CHOS) β-Mannanase (ManB) Glucomannan, Galactomannan Mannooligosaccharides (MOS) pSIP-based Expression Vectors OmpA Native Signal P Mature Enzyme Beta-Mannanase Chitosanase pSIP409 ery: erythromycin-resistant gene Lactobacillus plantarum WCFS1 OmpA Native Signal P Mature Enzyme Beta-Mannanase Chitosanase pSIP609 alr: alanine racemase gene Lactobacillus plantarum TLG02 (Δalr, D - alanine auxotroph) Nguyen et al, (2011). J Agric Food Chem 59, 5617-5624. Native signal peptide Csn OmpA signal peptide Csn pSIP609 (alr) Food-grade expression β-Mannanase (ManB) Rep1 Rep2 Rep3 E.coli Native signal peptide ManB OmpA signal peptide ManB 27700* 24500 53.1 % * 14200 (broth) + 13500 (periplasmic space) Conclusions • Two bacillus hydrolytic enzymes (Csn and ManB) were successfully expressed in L. plantarum food-grade expression system, using pSIP-based vector • Both E. coli OmpA and bacillus native signal peptides can direct the secretion of both enzymes into culture broth; however, native signal peptide is more efficient Kob-khun-ka Thank you Professor Vincent Eijsink (The Norwegian University of Life Sciences) NRU