Secretion of Bacillus Hydrolytic Enzymes using Food-grade...
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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
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Beta-mannanase, ManB
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Chitosanase, Csn
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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
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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 pH-
control
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pSIP-based expression system
Diep, D.B., Mathiesen, G., Eijsink, V.G., and Nes, I.F. (2009). Use of lactobacilli and their pheromone-based 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
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Using Alanine Racemase (alr) for Food-grade expression
D-alanine is a component of the peptidoglycan structure of bacterial cell wall alr
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Chitosanse (Csn)
Chitooligosaccharides (CHOS)
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β-Mannanase (ManB)
Glucomannan, Galactomannan
Mannooligosaccharides (MOS)
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pSIP-based Expression Vectors
pSIP409
Mature Enzyme Beta-Mannanase Chitosanase
OmpA Native Signal P
pSIP609
OmpA Native Signal P
Mature Enzyme Beta-Mannanase Chitosanase
ery: erythromycin-resistant gene alr: alanine racemase gene
Lactobacillus plantarum WCFS1 Lactobacillus plantarum TLG02 (Δalr, D - alanine auxotroph) Nguyen et al, (2011). J Agric Food Chem 59, 5617-5624.
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Csn
Csn
Native signal peptide
OmpA signal peptide
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pSIP609 (alr) Food-grade expression
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β-Mannanase (ManB)
ManB
ManB
Native signal peptide
OmpA signal peptide
Rep1
Rep2
Rep3
E.coli 27700* 24500 53.1 %
* 14200 (broth) + 13500 (periplasmic space)
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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
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Kob-khun-ka Thank you
Professor Vincent Eijsink (The Norwegian University of Life Sciences)
NRU