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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

42Northern analysis

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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