Applied and Environmental Microbiology, May 2006, p. 3448-3457
Transcript of Applied and Environmental Microbiology, May 2006, p. 3448-3457
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Aspergillus oryzae
Speaker: Chia-wen ChenAdvisor: Huang, C.-T2007/10/30
Applied and Environmental Microbiology, May 2006, p. 3448-3457, Vol. 72, No. 5
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Aspergillus oryzae
Rawlings, N.D., Morton, F.R. & Barrett, A.J. (2006) MEROPS: the peptidase database. Nucleic Acids Res 34, D270-D272.
Kingdom FungiPhylum AscomycotaSubphylum PezizomycotinaClass EurotiomycetesOrder EurotialesFamily TrichocomaceaeGenus AspergillusSpecies A. oryzae
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Aspergillus oryzaeGRAS (generally regarded as safe)A large amounts of enzymes
(mainly hydrolytic enzymes )
A wide range(glucoamylse a, alanyl dipeptidyl peptidase ……)
Commercial production (often extracellular)
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Solid-State Culture Submerged CultureNature environment Artificial environment
Often used in filamentous fungi Can be used in plant and animal cell
Low cost High cost
Hard to manipulate physical andchemical properties
Easy to manipulate physical andchemical properties
Purity low Purity high
Purify ?? Purify ☆
Food Pharmaceutical products
Low price High price
6http://www.vesuite.org/index.php
Solid-State CultureSubmerged Culture
http://www.unitus.it/dipartimenti/dabac/progetti/ssbioreactors/solidstatebioreactor.htm
pilot solid-state bioreactor
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glaB (glucoamylase B)pepA (alanyl dipeptidyl peptidase)Glycolytic pathway & TCA cycle
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Hydrolytic enzymes
A solid state-specific expression marker
Expression of glaB was also induced by starch ormalto-oligosacharides in surface cultures(Hata et al. 1998)
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Hydrolysis of X-Ala, His-Ser, and Ser-Tyrdipeptides at a neutral pH optimumThe encoding gene can’t be expressed insubmerged cultureCan be expressed when cultured onsteamed riceN-glycosylated.
(Kitano et al. 2002)
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Realize the mechanism causing the differencebetween 2 culture systems
throughtranscriptional analysisproteomic analysisESTs (Expressed Sequence Tags) ……
11ESTs are generated by sequencing cDNAUsually 200 to 500 nucleotides long
http://www.ncbi.nlm.nih.gov/About/primer/est.html
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Databasehttp://www.ncbi.nlm.nih.gov/projects/dbESTToolsBLAST, FASTA, Entrez, GenBank
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http://www.nrib.go.jp/ken/EST/db/index.html
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http://www.nrib.go.jp/ken/EST/db/blast.html
17http://www.bio.nite.go.jp/dogan/Top
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A comparative proteome analysis
Protein secretion profiles
Compare the difference between solid-stateand submerged culture
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Yield
Peptide massfingerprinting
MALDI-TOF MS
Incubate40 hours
Classifying proteins
HydrophobicChromatography
SDS-PAGE
Northern analysis
2-D gelelectrophoresis
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Dry mycelium weight →N- acetylglucosamineProtein concentration →
Bradford protein assay3 independent experiments
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The single unit of chitinChitin is the major component of fungi’s cellwall
http://www.ocean.udel.edu/horseshoecrab/Research/chitin.html
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10g 100 mL
Solid-State fermentation: Submerged fermentation=4-6.4 fold(protein secreted/gram of dry mycelium)
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Protein +Genome Proteome
Nature 405, 837-846(15 June 2000)
24Bakhtiar, R. et al. Mutagenesis 2000 15:415-430; doi:10.1093/mutage/15.5.415
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http://www.psrc.usm.edu/mauritz/maldi.html
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mH + hv → mH*mH* + M →m-+[M+H]+
http://www.psrc.usm.edu/mauritz/maldi.html
27http://qbab.aber.ac.uk/roy/mss/tofspec.htm
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http://www.expasy.org/
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http://www.expasy.org/tools/aldente/
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α-amylase
IPG:ph4-710-20% SDS polyacrylamide gel300 μgCBB stained
α-amylase α-amylase α-amylaseα-amylase
α-amylaseα-amylase α-amylase
Oryzin spotOnly appears in solid-state culture
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α-amylase
α-amylase α-amylase α-amylaseα-amylase
α-amylaseα-amylase α-amylase
Culture conditionsAge of the cultureSpots diffused-heterogeneity of proteins due to glycosylation
Wheat bran proteins degraded and vanished at 24 h
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Reason: N-linked oligosaccharides can bedifficult to see by MALDI-TOF MSMake peptides above 3500 kDaDeglycosylation of proteins before 2-D PAGE→protein insolubleA reasonable choice
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Cleave the β-aspartylglucosamine bond ofasparagine-linked oligosaccharides
http://www.rcsb.org/pdb/explore.do?structureId=1PNGhttp://www.glycoforum.gr.jp/science/word/glycoprotein/GPA00E.html
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http://www.rcsb.org/pdb/explore.do?structureId=2PTN
Trypsin Cleaves Exclusively C-terminal toArginine and Lysine Residues
http://gandalf.umd.edu/BCHM461/Exam2/Ex2_sol.pdf
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Solid-state culture Submerged culture
Cell wall-bound proteinin submerged culture
Incubate 40 hours10-20% SDS polyacrylamide gelCBB stainedIn-gel deglycosylated with PNGase F → trypsin digestionScores >60IPG:ph4-7
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Over 50% of visible proteins areα-amylases and their proteolytic products
37Glucoamylase A, Xylanase G2…….
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α-amylase and β-glucosidase
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Hydrophobic ChromatographyA280nm
α-amylase activity
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Solid state
Submerged state
Both conditions
Be trapped in cell wall
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Quantitative control
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Low external osmotic pressure in submergedculture→ a static cell wall→ protein can be trappedAspergillus kawachii can secrete α-amylaseand β-glucosidase → the help of extracellularpolysacharrides
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Submerged and Solid-StateCulture produce different
proteins.
We can divide proteins into 4 types.
Solid-State Cultureusually producemore proteins
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