Bacillus Enzymes using Food-grade Expression System

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
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